Yeast cells engineered to produce pheromone system protein surrogates, and uses therefor

ABSTRACT

Yeast cells are engineered to express both a surrogate of a pheromone system protein (e.g., enzymes involved in maturation of α-factor, transporters of a-factor, pheromone receptors, etc.) and a potential peptide modulator of the surrogate, in such a manner that the inhibition or activation of the surrogate affects a screenable or selectable trait of the yeast cells. Various additional features improve the signal-to-noise ratio of the screening/selection system.

This application is a continuation-in-part of Ser. No. 08/309,313, filedSep. 20, 1994, now abandoned, which is a continuation-in-part of Ser.No. 08/190,328, filed Jan. 31, 1994, now abandoned, which is acontinuation-in-part of Ser. No 08/041,431, filed Mar. 31, 1993, nowabandoned, all hereby incorporated by reference.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to the screening of drugs, especiallyrandom peptides, in yeast cells for the ability to interact withproteins involved in the post-translational modification, transport ofand response to yeast pheromones or substitutes therefor.

2. Description of the Background Art

Drug Screening

The identification of biological activity in new molecules hashistorically been accomplished through the use of in vitro assays orwhole animals. Intact biological entities, either cells or wholeorganisms, have been used to screen for anti-bacterial, anti-fungal,anti-parasitic and anti-viral agents in vitro. Cultured mammalian cellshave also been used in screens designed to detect potential therapeuticcompounds. A variety of bioassay endpoints are exploited in mammaliancell screens including the stimulation of growth or differentiation ofcells, changes in cell motility, the production of particularmetabolites, the expression of specific proteins within cells, alteredprotein function, and altered conductance properties. Cytotoxiccompounds used in cancer chemotherapy have been identified through theirability to inhibit the growth of tumor cells in vitro and in vivo Inaddition to cultures of dispersed cells, whole tissues have served inbioassays, as in those based on the contractility of muscle.

In vitro testing is a preferred methodology in that it permits thedesign of high-throughput screens: small quantities of large numbers ofcompounds can be tested in a short period of time and at low expense.Optimally, animals are reserved for the latter stages of compoundevaluation and are not used in the discovery phase; the use of wholeanimals is labor-intensive and extremely expensive.

Microorganisms, to a much greater extent than mammalian cells andtissues, can be easily exploited for use in rapid drug screens. Yeastprovide a particularly attractive test system; extensive analysis ofthis organism has revealed the conservation of structure and function ofa variety of proteins active in basic cellular processes in both yeastand higher eukaryotes.

The search for agonists and antagonists of cellular receptors has beenan intense area of research aimed at drug discovery due to the elegantspecificity of these molecular targets. Drug screening has been carriedout using whole cells expressing functional receptors and, recently,binding assays employing membrane fractions or purified receptors havebeen designed to screen compound libraries for competitive ligands. DukeUniversity, WO92/05244 (Apr. 2, 1992) describes the expression ofmammalian G protein-coupled receptors in yeast and a means ofidentifying agonists and antagonists of those receptors using thatorganism.

In addition, yeast are, of course, used in the discovery of antifungalcompounds; Etienne et al. (1990) describe the use of Saccharomycescerevisiae mutant strains, made highly sensitive to a large range ofantibiotics, for the rapid detection of antifungals.

Yeast Pheromone System Proteins and Their Metabolic Function

Haploid yeast cells are able not only to grow vegetatively, but also tomate to form a diploid cell. The two mating types ("sexes") of haploidcells are designated a and α. The a cells produce the dodecapeptidea-factor, and the α cells, the tridecapeptide α-factor. Because a-factorand α-factor elicit a mating response in the yeast cell of the opposite"sex", they are called "pheromones". These pheromones, as well as otherproteins specifically involved in the production or transport of, orresponse to, pheromones, are considered "pheromone system proteins".

The gene encoding a-factor pheromone, like the α-factor receptor gene,is an a cell-specific gene; a cell-specific genes are only expressed ina cells. The gene encoding α-factor pheromone, like the a-factorreceptor gene, is an α cell-specific gene; α cell-specific genes areonly expressed in α cells. Other yeast genes belong to ahaploid-specific gene set and are expressed in haploid cells (a cells orα cells) but not in diploid (a/α) cells. In addition, there exists adiploid cell-specific gene set, including those genes involved insporulation.

In eukaryotic cells, RNA polymerase II promoters contain a specificsequence (the TATA box) to which the transcription factor TFIID (TATAbinding protein or TBP) binds. An active transcription initiationcomplex includes TFIID, accessory initiation proteins, and RNA Pol II.As in higher eukaryotic cells, the TATA box is an essential controlsequence in yeast promoters. Yeast TATA-box-binding protein (TBP) wasidentified by its ability to substitute in function for mammalian TFIID[Buratowski et al., Nature 334, 37 (1988); Cavallini et al., Nature 334,77 (1988)]. With only a few apparent exceptions [transcription of someglycolytic enzyme genes, see Struhl, Mol. Cell. Biol. 6, 3847 (1986) andOgden et al., Mol. Cell Biol. 6, 4335 (1986)] transcription of yeastgenes requires the proximal TATA box element and TFIID binding forinitiation of transcription. Also required for efficient transcriptionare gene-specific activator proteins; the precise mechanism wherebythese gene-specific regulatory proteins influence transcription has notbeen completely elucidated.

MCM1p (encoded in the MCM1 gene) is a non-cell-type-specifictranscription factor in yeast. MCM1p acts alone or in concert with otherregulatory proteins to control expression of a- and α-cell specificgenes. Yeast mating type loci encode the regulatory proteins thatcontribute to the control of cell type-specific expression. Theseproteins are Mata1p (encoded by the MATa gene) and Matα1p and Matα2p(encoded by the MATα locus). MCM1p activates transcription of a-specificgenes by binding to an upstream activation sequence (UAS) located in thecontrol region of a-specific genes. Matα1p and MCM1p interact to enhanceeach other's binding to specific UAS binding sites to activateα-cell-specific gene transcription in α-cells. Matα2p associates withMCM1p to repress a-specific gene transcription in α-cells. In diploid(a/α) cells, Matα1p and Matα2p associate to repress the transcription ofhaploid-specific genes. The Matα1p/Matα2p regulatory entity is foundonly in diploid cells.

Yeast contain two genes encoding the α-factor pheromone, MFα1 and MFα2.Analysis of yeast bearing mutations in these sequences indicates thatMFα1 gives rise to the majority of α-factor produced by cells.Expression occurs at a higher level from MFα1 than from MFα2 (Kurjan,Mol. Cell. Biol. 5, 787 (1985). The MFα1 gene of yeast encodes a 165 aaprecursor protein containing an 85 aa leader sequence at the N-terminus.The leader includes a 19 aa signal sequence and a 66 aa sequence whichcontains sites for the addition of three oligosaccharide side chains(Kurjan and Herskowitz, Cell 39, 933 (1982); Singh et al. Nuc. AcidsRes. 11, 4049 (1983); Julius et al. Cell 36, 309 (1984). Four tandemcopies of the 13 aa α-factor are present in the C-terminal portion ofthe precursor; 6-8 aa spacer peptides precede the α-factor sequences(see FIG. 2).

After translocation of the nascent α-factor polypeptide to the ER, thesignal sequence is cleaved from the precursor protein to yieldpro-α-factor (Waters et al. J. Biol. Chem. 263, 6209 (1988). The coreN-linked carbohydrate is added to three sites in the N-terminus ofpro-α-factor (Emter et al. Biochem. Biophys. Res. Commun. 116, 822(1983); Julius et al. Cell 36, 309 (1984); Julius et al. Cell 37, 1075(1984). Additional glycosylation occurs in the Golgi prior to cleavageof pro-α-factor by the KEX2 endopeptidase. This enzyme cleaves withineach of the spacer repeats leaving a Lys-Arg sequence attached to theC-terminus of α-factor peptide (Julius et al. Cell 37, 1075 (1984). TheLys-Arg sequence is removed by the action of the KEX-1 protease(Dmochowska et al. Cell 50, 573 (1987). The additional spacer residuespresent at the N-terminus of α-factor peptide are removed by thedipeptidyl aminopeptidase encoded by STE13 (Julius et al. Cell 32, 839(1983). Four α-factor peptides are released from each precursor proteinvia the proteolytic processing outlined above and the mature α-factor issecreted from the cell.

Precursors of the 12 aa mature a-factor peptide are encoded in the MFa1and MFa2 genes and are 36 aa and 38 aa residues, respectively (forschematic of MFa1 gene see FIG. 5). The precursors contain one copy ofa-factor and the products of the two genes differ in sequence at oneamino acid. The two forms of a-factor are produced in equal amounts by acells (Manney et al. in Sexual interactions in eukaryotic microbes, p21,Academic Press, New York (1981).

Processing of a-factor entails a process that differs in every detailfrom that of α-factor. The processing of a-factor begins in the cytosoland involves the farnesylation of the C-terminal cysteine residue nearthe carboxyl terminus (-CVIA) by a farnesyl transferase (Schafer et al.Science 245, 379 (1989); Schafer et al. Science 249, 1133 (1990). The αand β subunits of the farnesyl transferase are encoded by the RAM2 andRAM1 genes, respectively (He et al. Proc. Natl. Acad. Sci. 88, 11373(1991). Subsequent to farnesylation is the proteolytic removal of thethree amino acids that are C-terminal to the modified cysteine by amembrane-bound endoprotease. Next, the carboxy-terminal farnesylatedcysteine residue is modified further: the carboxyl group is methylatedby the product of the STE14 gene. STE14p is a membrane-boundS-farnesyl-cysteine carboxyl methyl transferase (Hrycyna et al. EMBO. J.10, 1699 (1991). The mechanisms of the N-terminal processing of a-factorhave not been elucidated. After processing of the precursors iscomplete, mature a-factor is transported to the extracellular space bythe product of the STE6 gene (Kuchler et. al. EMBO J. 8, 3973 (1989), anATP-binding cassette (ABC) transporter.

In normal S. cerevisiae (budding yeast) a cells, the α-factor binds theG protein-coupled membrane receptor STE2. The G protein dissociates intothe G.sub.α and G.sub.βγ subunits, and the G.sub.βγ binds anunidentified effector, which in turn activates a number of genes. STE20,a kinase, activates STE5, a protein of unknown function. STE5 activatesSTE11 kinase, which stimulates STE7 kinase, which induces the KSS1and/or FUS3 kinases. These switch on expression of the transcriptionfactor STE12. STE12 stimulates expression of a wide variety of genesinvolved in mating, including FUS1 (cell fusion), FAR1 (cell-cyclearrest), STE2 (the receptor), MFA1 (the pheromone), SST2 (recovery),KAR3 (nuclear fusion) and STE6 (pheromone secretion). Other genesactivated by the pathway are CHS1 , AGα1, and KAR3 . The multiply tandemsequence TGAAACA has been recognized as a "pheromone response element"found in the 5'-flanking regions of many of the genes of this pathway.

One of the responses to mating pheromone is the transient arrest of theyeast cell in the G1 phase of the cell cycle. This requires that allthree G1 cyclins (CLN1, CLN2, CLN3) be inactivated. It is believed thatFUS3 inactivates CLN3, and FAR1 inhibits CLN2. (The product responsiblefor inactivating CLN1 is unknown).

The growth arrest is terminated by a number of different mechanisms.First, the α-factor receptor is internalized following binding of thepheromone, resulting in a transient decrease in the number of pheromonebinding sites. Second, the C-terminal tail of the receptor isphosphorylated consequent to ligand binding, resulting in uncoupling ofthe receptor from the transducing G proteins. Third, pheromone-inducedincreases in expression of GPA1p (the Gα-subunit of the heterotrimeric Gprotein) increase the level of the α subunit relative to the G.sub.β andG.sub.γ subunits, resulting in reduction in the level of free G.sub.βγand consequent inactivation of the pheromone response pathway.Additional mechanisms include induction of the expression of SST2 andBAR1 and phosphorylation of the α subunit (perhaps by SVG1).

Signaling is inhibited by expression of a number of genes, includingCDC36 , CDC39 , CDC72 , CDC73 , and SRM1. Inactivation of these genesleads to activation of the signaling pathway

A similar pheromone signaling pathway may be discerned in α cells, butthe nomenclature is different in some cases (e.g., STE3 instead of STE2).

Other yeast also have G protein-mediated mating factor responsepathways. For example, in the fission yeast S. pombe, the M factor bindsthe MAP3 receptor, or the P-factor the MAM2 receptor. The dissociationof the G protein activates a kinase cascade (BYR2, BYR1, SPK1), which inturn stimulates a transcription factor (STE11). However, in S. pombe,the Gα subunit transmits the signal, and there are of course otherdifferences in detail.

Pheromone Pathway Mutants

The effects of spontaneous and induced mutations in pheromone pathwaygenes have been studied. These include the α-factor (MFα1 and MFα2)genes, see Kurjan, Mol. Cell. Biol., 5:787 (1985); the a-factor (MFa1and MFa2) genes, see Michaelis and Herskowitz, Mol. Cell. Biol. 8:1309(1988); the pheromone receptor (STE2 and STE3 ) genes, see Mackay andManney, Genetics, 76:273 (1974), Hartwell, J. Cell. Biol., 85:811(1980), Hagen, et al., P.N.A.S. (U.S.A.), 83:1418 (1986); the FAR1 gene,see Chang and Herskowitz, Cell, 63:999 (1990); and the SST2 gene, seeChan and Otte, Mol. Cell. Biol., 2:11 (1982).

Expression of Foreign Proteins in Yeast Cells

A wide variety of foreign proteins have been produced in S. cerevisiae,either solely in the yeast cytoplasm or through exploitation of theyeast secretory pathway (Kingsman et al. TIBTECH 5, 53 (1987). Theseproteins include, as examples, insulin-like growth factor receptor(Steube et al. Eur. J. Biochem. 198, 651 (1991), influenza virushemagglutinin (Jabbar et al. Proc. Natl. Acad. Sci. 82, 2019 (1985), ratliver cytochrome P-450 (Oeda et al. DNA 4, 203 (1985) and functionalmammalian antibodies (Wood et al. Nature 314, 446 (1985). Use of theyeast secretory pathway is preferred since it increases the likelihoodof achieving faithful folding, glycosylation and stability of theforeign protein. Thus, expression of heterologous proteins in yeastoften involves fusion of the signal sequences encoded in the genes ofyeast secretory proteins (e.g., α-factor pheromone or the SUC2[invertase] gene) to the coding region of foreign protein genes.

A number of yeast expression vectors have been designed to permit theconstitutive or regulated expression of foreign proteins. Constitutivepromoters are derived from highly expressed genes such as those encodingmetabolic enzymes like phosphoglycerate kinase (PGK1) or alcoholdehydrogenase I (ADH1) and regulatable promoters have been derived froma number of genes including the galactokinase (GAL1) gene. In addition,supersecreting yeast mutants can be derived; these strains secretemammalian proteins more efficiently and are used as "production" strainsto generate large quantities of biologically active mammalian proteinsin yeast (Moir and Davidow, Meth. in Enzymol. 194, 491 (1991).

A variety of heterologous proteins have been expressed in yeast cells asa means of generating the quantity of protein required for commercialuse or for biochemical study (Kingsman et al. TIBTECH 5, 53 (1987). Inaddition, a number of mammalian proteins have been expressed in yeast inorder to determine whether the proteins (1) will functionally substitutefor cognate proteins normally expressed within that organism or (2) willinteract with accessory yeast proteins to accomplish a specificfunction. Thus it has been determined that a human TBP with alteredbinding specificity will function to initiate transcription in yeast[Strubin and Struhl, Cell 68, 721 (1992)]. In addition, mammaliansteroid hormone receptors [Metzger et al. (1988); Schena and Yamamoto(1988)] and human p53 [Scharer and Iggo, Nuc. Acids Res. 20, 1539(1992)] were shown to activate transcription in yeast.

Expression in yeast of the gag-pol gene of HIV-1 results in theprocessing of the gag protein precursor to yield the products whichnormally arise within the virion; processing in yeast, as in the virus,is due to the action of the protease encoded within the gag-pol gene(Kramer et al. Science 231,1580 (1986).

A number of mammalian ABC transporters have been expressed in yeast todetermine their ability to substitute for yeast Ste6p in the transportof pheromone. The mammalian proteins thus far tested include human Mdr1(Kuchler and Thorner, Proc. Natl. Acad. Sci. 89, 2302 (1992)) and murineMdr3 (Raymond et al. Science 256, 232 (1992), proteins involved inmultidrug resistance; in addition, a chimeric protein containing humanCFTR (cystic fibrosis transmembrane conductance regulator) and yeastSTE6 sequence has been shown to transport a-factor pheromone in yeast(Teem et al. Cell 73, 335 (1993).

An a cell may be engineered to produce the a-factor receptor, and an αcell to make α-factor receptor. Nakayama, et al., EMBO J., 6:249-54(1987); Bender and Sprague, Jr., Genetics 121: 463-76 (1989).

Heterologous G protein-coupled receptors have been functionallyexpressed in S. cerevisiae. Marsh and Hershkowitz, Cold Spring HarborSymp., Quant. Biol., 53: 557-65 (1988) replaced the S. cerevisiae STE2with its homologue from S. Kluyven. More dramatically, a mammalianbeta-adrenergic receptor and Gα subunit have been expressed in yeast andfound to control the yeast mating signal pathway. King, et al., Science,250: 121-123 (1990).

Duke University, WO92/05244 (Apr. 2, 1992) describes a transformed yeastcell which is incapable of producing a yeast G protein α subunit, butwhich has been engineered to produce both a mammalian G protein αsubunit and a mammalian receptor which is "coupled to" (i.e., interactswith) the aforementioned mammalian G protein α subunit. Specifically,Duke reports expression of the human beta-2 adrenergic receptor (hβAR),a seven transmembrane receptor (STR), in yeast, under control of theGAL1 promoter, with the hβAR gene modified by replacing the first 63base pairs of coding sequence with 11 base pairs of noncoding and 42base pairs of coding sequence from the STE2 gene. (STE2 encodes theyeast α-factor receptor). Duke found that the modified hβAR wasfunctionally integrated into the membrane, as shown by studies of theability of isolated membranes to interact properly with various knownagonists and antagonists of hβAR. The ligand binding affinity foryeast-expressed hβAR was said to be nearly identical to that observedfor naturally produced hβAR.

Duke co-expressed a rat G protein α subunit in the same cells, yeaststrain 8C, which lacks the cognate yeast protein. Ligand bindingresulted in G protein-mediated signal transduction.

Duke teaches that these cells may be used in screening compounds for theability to affect the rate of dissociation of Gα from Gβγ in a cell. Forthis purpose, the cell further contains a pheromone-responsive promoter(e.g. BAR1 or FUS1 ), linked to an indicator gene (e.g. HIS3 or LacZ).The cells are placed in multi-titer plates, and different compounds areplaced in each well. The colonies are then scored for expression of theindicator gene.

Duke's yeast cells do not, however, actually produce the compounds to bescreened. As a result, only a relatively small number of compounds canbe screened, since the scientist must ensure that a given group of cellsis contacted with only a single, known compound.

Yeast have been engineered to express foreign polypeptide variants to betested as potential antagonists of mammalian receptors. Librariesencoding mutant glucagon molecules were generated through randommisincorporation of nucleotides during synthesis of oligonucleotidescontaining the coding sequence of mammalian glucagon. These librarieswere expressed in yeast and culture broths from transformed cells wereused in testing for antagonist activity on glucagon receptors present inrat hepatocyte membranes (Smith et al. 1993).

Drugs which overcome the multiple drug resistance (MDR) of cancer cellsmay be identified by using transformed yeast cells expressingP-glycoprotein (Suntory Ltd., patent application JP 2257873 entitled"Multiple drug resistance-relating gene-comprises P-glycoproteinaccumulated in cell membrane part of transformed yeast"). The drugs werenot produced by the yeast cells in question.

A yeast strain has been derived to allow the identification ofinhibitors of protein farnesyltransferase which exhibit activity againstmammalian Ras and which may therefore function as antitumor drugs (Haraet al. 1993)

Genetic Markers in Yeast Strains

Yeast strains that are auxotrophic for histidine (HIS3) are known, seeStruhl and Hill), Mol. Cell. Biol., 7:104 (1987); Fasullo and Davis,Mol. Cell. Biol., 8:4370 (1988). The HIS3 (imidazoleglycerol phosphatedehydratase) gene has been used as a selective marker in yeast. SeeSikorski and Heiter, Genetics, 122:19 (1989); Struhl, et al., P.N.A.S.76:1035 (1979); and, for FUS1-HIS3fusions, see Stevenson, et al., GenesDev., 6:1293 (1992).

Peptide Libraries

Peptide libraries are systems which simultaneously display, in a formwhich permits interaction with a target, a highly diverse and numerouscollection of peptides. These peptides may be presented in solution(Houghten), or on beads (Lam), chips (Fodor), bacteria (Ladner U.S. Pat.No. 5,223,409), spores (Ladner U.S. Pat. No. '409), plasmids (Cull) oron phage (Scott, Devlin, Cwirla, Felici, Ladner U.S. Pat. No. '409).Many of these systems are limited in terms of the maximum length of thepeptide or the composition of the peptide (e.g., Cys excluded). Stericfactors, such as the proximity of a support, may interfere with binding.Usually, the screening is for binding in vitro to an artificiallypresented target, not for activation or inhibition of a cellular signaltransduction pathway in a living cell. While a cell surface receptor maybe used as a target, the screening will not reveal whether the bindingof the peptide caused an allosteric change in the conformation of thereceptor.

Ladner, U.S. Pat. No. 5,096,815 describes a method of identifying novelproteins or polypeptides with a desired DNA binding activity.Semi-random ("variegated") DNA encoding a large number of differentpotential binding proteins is introduced, in expressible form, intosuitable host cells. The target DNA sequence is incorporated into agenetically engineered operon such that the binding of the protein orpolypeptide will prevent expression of a gene product that isdeleterious to the gene under selective conditions. Cells which survivethe selective conditions are thus cells which express a protein whichbinds the target DNA. While it is taught that yeast cells may be usedfor testing, bacterial cells are preferred. The interactions between theprotein and the target DNA occur only in the cell (and then only in thenucleus), not in the periplasm or cytoplasm, and the target is a nucleicacid, and not a pheromone system protein surrogate.

Substitution of random peptide sequences for functional domains incellular proteins permits some determination of the specific sequencerequirements for the accomplishment of function. Though the details ofthe recognition phenomena which operate in the localization of proteinswithin cells remain largely unknown, the constraints on sequencevariation of mitochondrial targeting sequences and protein secretionsignal sequences have been elucidated using random peptides (Lemire etal., J. Biol. Chem. 264, 20206 (1989) and Kaiser et al. Science 235, 312(1987), respectively).

All references cited in this specification are hereby incorporated byreference. No admission is made that any reference constitutes priorart.

SUMMARY OF THE INVENTION

In the present invention, a yeast cell is engineered to express anexogenous protein which is, however, capable of substituting for a yeastprotein which is involved in the post-translational modification,transport, recognition or signal transduction of a yeast pheromone,sufficiently, to be able, at least under some circumstances, to carryout that role of the yeast protein. For the sake of convenience, theseyeast proteins will be referred to as "pheromone system proteins" (PSP),and their cognate non-yeast proteins as PSP surrogates.

The pheromone system of a yeast cell is thus subverted so that theresponse of the cell to a yeast pheromone is at least partiallydetermined by the activity of the surrogate PSP. In a preferredembodiment, the cognate yeast PSP is not produced in functional form, sothat the response is essentially entirely dependent on the activity ofthe surrogate PSP.

Such yeast cells may be used to identify drugs which inhibit oractivate, to a detectable degree, the ability of the surrogate tosubstitute for the cognate yeast PSP. To screen for an inhibitor, anormally functional surrogate is expressed, and the presence of aninhibitor is indicated by a depression of the cellular response. Toscreen for an activator, a surrogate functional in yeast, or onenormally not functional in yeast but which is activatable (the latter ispreferred, to minimize background) is used, and the activator isdetected through its elevation of the cellular response.

In a preferred embodiment, the candidate drug is a peptide, and theyeast cell is engineered to express the candidate drug as well as thesurrogate PSP.

Another consideration is that with wild-type yeast cells, to achievepheromone secretion and response, both α and a cells must be provided.In some preferred embodiments, α cells are engineered to expressα-factor receptor or a cells are engineered to express a-factorreceptor. These modified cells may be considered "autocrine" becausethey are "self-stimulatory". Cells which express both a surrogate forthe pheromone receptor, and a heterologous peptide agonist for thesurrogate receptor, are also considered "autocrine", because they willrespond to the co-produced agonist.

The classes of PSPs are numerous. However, some examples may be helpful.

Farnesyltransferases and carboxymethyltransferases

After expression, a-factor is farnesylated by RAM1p and RAM2p andcarboxymethylated by Ste14p. These modifications are required foractivity.

RAM1p and RAM2p are homologous to the subunits of the heterodimericmammalian farnesyltransferase, which itself is necessary for membraneassociation of mammalian Ras proteins. If a yeast cell is engineered toexpress the mammalian farnesyltransferase, it may be used to identifydrugs which interact with that enzyme by determining whether afunctional a-factor is produced. Similarly, Ste14p is homologous tomammalian carboxymethyltransferases, which play regulatory roles incontrolling the function of low molecular weight G proteins (Ras, Rho,Rab).

Proteases

The PSP may be a yeast protease, such as KEX2, STE13 or KEX1. Yeastα-factor pheromone is generated through the controlled and limitedproteolysis of precursor proteins by these proteases. A yeast cell maybe engineered to express an inactive precursor of yeast α-factor inwhich a peptide linker, corresponding to the cleavage site of asurrogate non-yeast protease, is incorporated so that cleavage willliberate mature α-factor (or its functional homologue). For example, thePSP surrogate may be HIV protease, with the cleavage site of HIVprotease being substituted for the yeast protease cleavage sites in theα-factor precursor. The precursor and the HIV protease are co-expressedin the yeast cell. Proteolysis by HIV protease will give rise toproduction of mature α-factor and initiation of signal transduction.This system may be used to identify inhibitors of HIV protease.

Preferably, unlike yeast cells occurring in nature, the yeast cell isengineered not only to express the α-factor precursor, but also theα-factor receptor, so that a single haploid type of yeast is all that isrequired to conduct the assay.

ABC Transporters

Ste6 is the yeast ABC transporter necessary for the export of a-factor.The yeast cell is engineered to express both a-factor and a foreign ABCtransporter. This transporter may be one which is not, by itself, ableto transport a-factor, but which in the presence of a drug of interest,is capable of doing so, or it may be one which is already functional.

Preferably, the yeast cell is engineered to express not only a-factor,but also the a-factor receptor.

G Protein-Coupled Receptors

The PSP may be a yeast pheromone receptor. The surrogate is a non-yeast,G protein-coupled receptor. In order to achieve coupling to thepheromone signal transduction pathway, it may be necessary to provide aforeign or chimeric G.sub.α or G.sub.βγ subunit.

The engineered yeast cell may be used to screen for agonists as well asantagonists. When used to screen for agonists, it is preferable that theyeast pheromone not be produced in functional form.

Protein Kinases

The PSP may be a protein kinase, such as the FUS1, KSS1, STE11 or STE7proteins, which participate in the cellular response to pheromones. ThePSP surrogate would be, e.g., a mammalian, mitogen-activated proteinkinase. Yeast cells engineered to express the surrogate protein kinasecould be used to screen for activators or inhibitors thereof.

Cyclins

The PSP may be a cyclin, such as CLN1, CLN2 or CLN3. The cyclinsregulate the progression of cells through the cell cycle. The human C,D1 and E cyclins are capable of substituting functionally for the CLNproteins of yeast. Inhibitors of mammalian cyclins may be useful incancer chemotherapy. Yeast cells engineered to express a surrogatecyclin may be used to identify molecules which inhibit (or enhance) itsactivity.

Peptide Libraries

While others have engineered yeast cells to facilitate screening ofexogenous drugs as receptor agonists and antagonists, the cells did notthemselves produce both the drugs and the receptors. Yeast cellsengineered to produce the receptor, but that do not produce the drugsthemselves, are inefficient. To utilize them one must bring a sufficientconcentration of each drug into contact with a number of cells in orderto detect whether or not the drug has an action. Therefore, a microtiterplate well or test tube must be used for each drug. The drug must besynthesized in advance and be sufficiently pure to judge its action onthe yeast cells. When the yeast cell produces the drug, the effectiveconcentration is higher.

In a preferred embodiment, the yeast cells collectively produce a"peptide library", preferably including at least 10⁷ different peptides,so that diverse peptides may be simultaneously assayed for the abilityto interact with the PSP surrogate.

In an especially preferred embodiment, at least some peptides of thepeptide library are secreted into the periplasm, where they may interactwith the "extracellular" binding site(s) of an exogenous receptor. Theythus mimic more closely the clinical interaction of drugs with cellularreceptors. This embodiment optionally may be further improved (in assaysnot requiring pheromone secretion) by preventing pheromone secretion,and thereby avoiding competition between the peptide and the pheromonefor signal peptidase and other components of the secretion system.

Detection of Signal Transduction

Yeast cells may also be engineered so that their pheromone signaltransduction pathways provide a more readily detectable evidence of theactivity of a suspected drug. In these embodiments, the drug need not bea peptide produced by the same yeast cell, or even a peptide at all.

As previously mentioned, one of the consequences of activation of thepheromone signal pathway in wild-type yeast is growth arrest. If one istesting for an antagonist of a G protein-coupled receptor, or of otherpheromone system proteins, this normal response of growth arrest can beused to select cells in which the pheromone response pathway isinhibited. That is, cells exposed to both a known agonist and a peptideof unknown activity will be growth arrested if the peptide is neutral oran agonist, but will grow normally if the peptide is an antagonist.Thus, the growth arrest response can be used to advantage to discoverpeptides that function as antagonists.

However, when searching for peptides which can function as agonists of Gprotein-coupled receptors, or other pheromone system proteins, thegrowth arrest consequent to activation of the pheromone response pathwayis an undesirable effect for this reason: cells that bind peptideagonists stop growing while surrounding cells that fail to bind peptideswill continue to grow. The cells of interest, then, will be overgrown ortheir detection obscured by the background cells, confoundingidentification of the cells of interest. To overcome this problem thepresent invention teaches engineering the cell such that: 1) growtharrest does not occur as a result of pheromone signal pathway activation(e.g., by inactivating the FAR1 gene); and/or 2) a selective growthadvantage is conferred by activating the pathway (e.g., by transformingan auxotrophic mutant with a HIS3 gene under the control of apheromone-responsive promoter, and applying selective conditions).

It is, of course, desirable that the exogenous receptor (or other PSPsurrogate) be exposed on a continuing basis to the peptides.Unfortunately, this is likely to result in desensitization of thepheromone pathway to the stimulus. Desensitization may be avoided bymutating (which may include deleting) the SST2 gene so that it no longerproduces a functional protein, or by mutating other genes which maycontribute to desensitization, e.g., BAR1 in the case of a cells andSVG1 for either a or α cells.

If the endogenous pheromone receptor (or other cognate PSP) is producedby the yeast cell, the assay will not be able to distinguish betweenpeptides which interact with the pheromone receptor (or other cognatePSP) and those which interact with the exogenous receptor (or other PSPsurrogate). It is therefore desirable that the endogenous gene bedeleted or otherwise rendered nonfunctional.

The claims are hereby incorporated by reference as a further descriptionof the preferred embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Outline of successive stages in the development of yeastautocrine systems.

An outline of the normal synthesis and release of mating pheromones isdiagrammed in the upper left. Two genes, MFα1 and MFα2, encode precursorproteins (MFα1p and MFα2p) containing four and two repeats,respectively, of the tridecapeptide representing mature α-factor. Theseprecursors are processed proteolytically in a series of enzymaticreactions that begin with cleavage of the signal sequence in theendoplasmic reticulum and involve both glycosylation of the leaderpeptide and cleavage by the proteases KEX2p, STE13p, and KEX1P. Theresult is the secretion of mature α-factor which, upon binding to STE2pnormally expressed on the surface of a cells, elicits a number ofchanges in the a cells, including growth arrest. The a cells, in turn,express two genes, MFa1 and MFa2, which encode precursors (MFa1p andMFa2p) for a-factor. These precursors undergo farnesylation by RAM1 andRAM2, proteolytic trimming of the C-terminal three amino acids (by aprotein tentatively identified as RAM3p), carboxymethylation of thenewly exposed C-terminal cysteine by STE14p, and endoproteolytic removalof the N-terminal leader sequence by an activity provisionallyidentified as STE19p. Upon export of the mature a-factor from the cellvia STE6p, it binds to STE3p expressed on the surface of α cells andstops their growth.

Stage 1 involves the development of yeast strains in which SST2, FAR1,and HIS3 are inactivated and a suitable reporter construct likefus1::HIS3 is integrated into the genomes of both α and a cells. α cellsare further altered by replacement of the normally expressed STE3p withSTE2p, while a cells are further modified by replacement of the normallyexpressed STE2p with STE3p. The resulting strains should show growth onhistidine-deficient media in the absence of exogenous pheromone.

Stage 2 involves, first, inactivation of MFα1 and MFα2 in cells andinactivation of MFa1 and MFa2 in a cells developed in Stage 1. Thesemodifications will result in strains which are auxotrophic forhistidine. Next, the appropriate expression plasmid will be introduced:the expression plasmid pADC-MF (see FIG. 4) containing anoligonucleotide encoding α-factor should confer upon α cells the abilityto grow on histidine-deficient media; the expression plasmid pADC-MFa(see FIG. 6) containing an oligonucleotide encoding a-factor shouldenable a cells to grow on histidine-deficient media.

Stage 3 uses the cells developed in Stage 2 for the insertion ofexpression plasmids. However, instead of using plasmids which containoligonucleotides that encode genuine pheromone, the yeast will betransformed with expression plasmids that contain random or semi-randomoligonucleotides. Transformants which can grow on histidine-deficientmedia will be expanded and their plasmids isolated for sequencing of theinserted oligonucleotide.

FIG. 2. Diagram of the plasmid used for mutagenesis of MFα1. A 1.8 kbEcoRI fragment containing MFα1 is cloned into the EcoRI site of pALTERsuch that single-stranded DNA containing the MFα1 minus strand can besynthesized. The diagram illustrates the different regions of MFα1,including the promoter, transcription terminator, and different domainsof the precursor protein: the signal peptide, the pro peptide, the fourrepeats of mature α-factor, and the three spacers which separate theserepeats. Above the block diagram of the regions of MFα1 are the aminoacid sequences (SEQ ID NO:1) of the signal peptide and the pro peptide;below it are those of the pheromone repeats and the spacers (SEQ IDNO:2). The sites of proteolytic processing of the precursor protein areindicated by arrows, with each proteolytic activity represented by adifferent arrow, as indicated in the figure.

FIG. 3. Diagram of the plasmids used in the construction of the MFαexpression cassette. pAAH5 contains the ADC1 promoter which will be usedto drive expression of synthetic oligonucleotides inserted into the MFαexpression cassette. The 1.5 kb BamHI to HindIII fragment containing theADC1 promoter will be cloned into pRS426, a plasmid which functions as ahigh-copy episome in yeast, to yield pRS-ADC. pRS-ADC will be therecipient of MFα1 sequences which have been mutated as follows: theregion of MFα1 which encodes mature α-factor will be replaced withrestriction sites that can accept oligonucleotides with AflII and BglIIends. Insertion of oligonucleotides with AflII and BglII ends will yielda plasmid which encodes a protein containing the MFα1 signal and leadersequences upstream of the sequence encoded by the oligonucleotide. TheMFα1 signal and leader sequences should direct the processing of thisprecursor protein through the pathway normally used for the secretion ofmature α-factor.

FIG. 4. Diagram of constructs used for the expression of randomoligonucleotides in the context of MFα1. Oligonucleotides containing aregion of 39 random base pairs (shown at the top of the figure) will becloned into the AflII and BglII sites of the MFα1 expression cassette.These oligonucleotides will encode the six amino acids immediatelyN-terminal to the first repeat of the α-factor in MFα1, followed insuccession by a tridecapeptide of random sequence and a stop codon.Yeast transformed with these constructs and selected for an ability togrow on media deficient in uracil will use the ADC1 promoter to expressa protein consisting of the MFα1 leader (both pre and pro peptides)followed by 13 random amino acids. Processing of the leader sequenceswill result in secretion of the tridecapeptide. A nucleotide sequence(SEQ ID NO:3) is presented upstream of the leader sequence and a secondnucleotide sequence (SEQ ID NO:4) containing AflII, XhoI and BglII sitesand coding for a peptide with an amino acid sequence (SEQ ID NO:5) ispresented downstream from the leader sequence. A nucleotide sequence(SEQ ID NO:6) containing an AflII and a BclI site encodes for a peptidewith an amino acid sequence (SEQ ID NO:7).

FIG. 5. Diagram of the plasmid used for mutagenesis of MFa1. A 1.6 kbBamHI fragment containing MFa1 is cloned into the BamHI site of PALTERsuch that single-stranded DNA containing the MFa1 minus strand can besynthesized. The diagram illustrates the different regions of MFa1,including the promoter, transcription terminator, and different domainsof the precursor protein: the leader peptide; the dodecapeptide thatrepresents the peptide component of mature a-factor and whose C-terminalcysteine becomes farnesylated and carboxymethylated during processing;and the C-terminal three amino acids that are removed during processingof the precursor. Above the block diagram of the regions of MFa1 is theamino acid sequence (SEQ ID NO:8) of the primary translation product.

FIG. 6. Diagram of constructs used for the expression of randomoligonucleotides in the context of MFa1. Oligonucleotides containing aregion of 33 random base pairs (shown at the top of the figure) will becloned into the XhoI and AflII sites of the MFa1 expression cassette.These oligonucleotides will encode the seven amino acids immediatelyN-terminal to the first amino acid of mature a-factor, followed insuccession by a monodecapeptide of random sequence, a cysteine which isfarnesylated and carboxymethylated during processing of the precursor,three amino acids (VIA) which are proteolytically removed duringprocessing, and a stop codon. Yeast transformed with these constructsand selected for an ability to grow on media deficient in uracil willuse the ADC1 promoter to express a precursor protein consisting of theMFa1 leader followed by 11 random amino acids and a C-terminaltetrapeptide CVIA. Processing of this precursor will result in secretionof a C-terminally farnesylated, carboxymethylated dodecapeptide whichconsists of 11 random amino acids and a C-terminal cysteine. Anucleotide sequence (SEQ ID NO:9) is presented upstream of the MFaIleader and a second nucleotide sequence (SEQ ID NO:10) containing anAflII and a XhoI site and encoding for a peptide with an amino acidsequence (SEQ ID NO:11) is presented downstream from the MFaI leader. Anucleotide sequence (SEQ ID NO:12) containing corresponding AflII andXhoI sites for cloning into SEQ ID NO:10 encodes for a peptide with anamino acid sequence (SEQ ID NO:13).

FIG. 7. Autocrine Mata strain secretes and responds to signalling byα-factor.

A synthetic oligonucleotide encoding the yeast α-factor pheromone wasexpressed in Mata cells. These cells normally express the a-factorpheromone but were prevented from doing so by deletion of the endogenousa-factor-encoding genes. Expression and release of α-factor by thesecells renders them "autocrine" with regard to pheromone signalling. Thepeptide containing mature α-factor was processed within these cells fortransport through the yeast secretory pathway to the extracellularenvironment. Pheromone signalling was initiated by the binding ofα-factor to the Ste2 receptor expressed in Mata cells. Signalling bypheromone in the strain background used in these experiments results ingrowth of responsive cells on media that is deficient in histidine.Background growth of control cells that are not expressing α-factor isprevented by increasing concentrations of the HIS3 inhibitor,aminotriazole.

FIG. 8. Autocrine MATa strain secretes and responds to signalling bya-factor.

Yeast a-factor was expressed from a plasmid containing a syntheticpheromone-encoding oligonucleotide in Mata cells. The yeast used inthese experiments were made "autocrine" by replacement of the normallyexpressed Ste2 protein with the receptor for a-factor, Ste3. Thea-factor peptide was processed in these cells and transported to theextracellular environment by the endogenous Ste6 protein, anATP-dependent transmembrane transporter. Pheromone signalling initiatedby the a-factor released by these cells when bound to Ste3 is indicatedby the growth of the cells on histidine-deficient media. Backgroundgrowth of control cells, which are incapable of expressing a-factor(these α cells lack the plasmid which encodes the pheromone) isprevented by increasing concentrations of the HIS3 inhibitor,aminotriazole.

FIG. 9. Plasmid pYMA177 containing mutant human MDR1 (G185V mutation).

The plasmid pYMA177 was constructed by Karl Kuchler and permits thesimultaneous overexpression of both a mutant human Mdr1 protein and theyeast a-factor pheromone precursor (Kuchler & Thorner, Proc. Natl. Acad.Sci. 89, 2302 (1992) Cadus 1270, containing a galactose inducible,amino-terminal myc-tagged form of the C5a receptor, was used totransform a protease deficient strain of yeast.

FIG. 10. Activity of a fus1 promoter in response to signalling by humanC5a expressed in autocrine strains of yeast.

To verify and quantify pheromone pathway activation upon stimulation ofthe C5a receptor by C5a in yeast, the activity of the fus1 promoter wasdetermined colorometrically using a fus1-lacZ fusion. CY878 (MATα tbt1-1fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156 gpa1 (41)-Gαi2) wastransformed with CADUS 1584 (pRS424-fus1-lacZ) in addition to receptorand ligand plasmids listed below. Transformants were grown overnight insynthetic medium lacking leucine, uracil, and tryptophan, pH 6.8, 50 mMPIPES to an OD₆₀₀ of less than 0.8 and β-galactosidase activity(Guarente 1983) was assayed.

Cadus 1289+Cadus 1215=Receptor⁻ Ligand⁻ =(R-L-)

Cadus 1303+Cadus 1215=Receptor⁺ Ligand⁻ =(R+L-)

Cadus 1289+Cadus 1297=Receptor⁻ Ligand⁺ =(R-L+)

Cadus 1303+Cadus 1297=Receptor+ Ligand⁺ =(R+L+)

Receptor refers to the human C5a receptor.

Ligand refers to human C5a.

FIG. 11. This Figure schematically describes three hybrids of GPA1 andGαS. The -LLLLGAGES- sequence demarcated in GPA1 directly follows thenon-conserved N-terminal domain of the protein. The longer sequencedemarcated in GPA1 encodes the "switch region" believed to be involvedin the conformational change that occurs with nucleotide exchange uponreceptor activation. 41-Gαs is comprised of the N-terminal 41 aminoacids of GPA1 l linked to Gαs sequence from which the native N-terminalsequence has been deleted. SGS denotes a molecule comprised of theswitch region residues of GPA1 replacing those of Gαs. GPA₄₁ -SGSincludes both the N-terminal and switch region sequences of GPA1inserted into Gαs. (See Table 6 for the exact sequence junctions used toconstruct these hybrid proteins).

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention contemplates the assaying of peptides, especiallywhen presented in peptide libraries, expressed in genetically engineeredyeast cells, for the ability of the peptides to interact with pheromonesystem proteins and PSP surrogates produced by those yeast cells.

For the purpose of the present invention, an "exogenous" protein is onewhich sufficiently differs in amino acid sequence from the proteinsnaturally produced by the yeast cell in question so that its closestcognate is a protein produced by a cell other than a yeast cell. Thecell producing this cognate protein may be a microbial cell (other thana yeast cell), a plant cell, or an animal cell. If an animal cell, itmay be of invertebrate (e.g., insect or nematode) or of vertebrate(e.g., avian, piscine or mammalian, especially human) origin. A proteinis considered to be of, e.g., human origin, regardless of whether it isencoded by the chromosome of a normal human, or by the genome of a viruswhich infects and replicates in human cells.

An "activator" of a pheromone system protein surrogate is a substancewhich, in a suitable yeast cell, causes the pheromone system proteinsurrogate to become more active, and thereby elevates the signaltransduced by the native or modified pheromone signal pathway of saidcell to a detectable degree. The surrogate may be initiallynonfunctional, but rendered functional as a result of the action of theactivator, or it may be functional, and the effect of the activator isto heighten the activity of the surrogate. The mode of action of theactivator may be direct, e.g., through binding the surrogate, orindirect, e.g., through binding another molecule which otherwiseinteracts with the surrogate. When the PSP surrogate is a substitute fora pheromone receptor, and the activator takes the place of thepheromone, it is customary to refer to the activator as an agonist ofthe receptor.

Conversely, an "inhibitor" of a pheromone system protein surrogate is asubstance which, in a suitable yeast cell, causes the PSP surrogate tobecome less active, and thereby reduces the transduced signal to adetectable degree. The reduction may be complete or partial. When thePSP surrogate is a substitute for a pheromone receptor, and theinhibitor competes with the pheromone for binding to the receptor, it iscustomary to refer to the inhibitor as an "antagonist".

The term "modulator" includes both "activators" and "inhibitors".

A surrogate PSP protein is "functionally homologous" to a yeast proteinif, either alone or after being modified by a drug, it is able toperform the function of the yeast PSP, or an analogous function, withinthe engineered yeast cell. It is not necessary that it be as efficientas the yeast protein, however, it is desirable that it have at least 10%of at least one of the pheromone system-related activities of the yeastprotein. Nor is it necessary that it have the same spectrum of action asthe yeast protein, e.g., if it is a receptor, it may respond to entirelydifferent ligands than does the endogenous receptor, or to some commonligands and some new ones. The receptors of Table 2 are consideredfunctionally homologous with the yeast pheromone receptors, even thoughthey do not respond to yeast pheromones, and may not couple to theunmodified endogenous G proteins, although they are G protein-coupledreceptors. This is considered an "analogous function".

The PSP surrogate may be a protein which must be modified in some way bya drug to be functional. For example, the drug could cause an allostericchange in the PSP surrogate's conformation, or it could cleave off aportion of the surrogate, the balance of the protein then being afunctional molecule.

The PSP surrogate may also be one which is functional only if othermodifications are made in the yeast cell, e.g., expression of a chimericG α subunit to interact with an exogenous G protein-coupled receptor.

The term "substantially homologous", when used in connection with aminoacid sequences, refers to sequences which are substantially identical toor similar in sequence, giving rise to a homology in conformation andthus to similar biological activity. The term is not intended to imply acommon evolution of the sequences.

Typically, "substantially homologous" sequences are at least 50%, morepreferably at least 80%, identical in sequence, at least over anyregions known to be involved in the desired activity. Most preferably,no more than five residues, other than at the termini, are different.Preferably, the divergence in sequence, at least in the aforementionedregions, is in the form of "conservative modifications".

"Conservative modifications" are defined as

(a) conservative substitutions of amino acids as hereafter defined; and

(b) single or multiple insertions or deletions of amino acids at thetermini, at interdomain boundaries, in loops or in other segments ofrelatively high mobility.

Preferably, except at the termini, no more than about five amino acidsare inserted or deleted at a particular locus, and the modifications areoutside regions known to contain binding sites important to activity.

Conservative substitutions are herein defined as exchanges within one ofthe following five groups:

I. Small aliphatic, nonpolar or slightly polar residues:

Ala, Ser, Thr (Pro, Gly)

II. Polar, negatively charged residues: and their amides

Asp, Asn, Glu, Gln

III. Polar, positively charged residues:

His, Arg, Lys

IV. Large, aliphatic, nonpolar residues:

Met, Leu, Ile, Val (Cys)

V. Large, aromatic residues:

Phe, Tyr, Trp

Residues Pro, Gly and Cys are parenthesized because they can havespecial conformational roles. Cys participates in formation of disulfidebonds. Gly imparts flexibility to the chain. Pro imparts rigidity to thechain and disrupts α helices. These residues may be essential in certainregions of the polypeptide, but substitutable elsewhere.

Two regulatory DNA sequences (e.g., promoters) are "substantiallyhomologous" if they have substantially the same regulatory effect as aresult of a substantial identity in nucleotide sequence. Typically,"substantially homologous" sequences are at least 50%, more preferablyat least 80%, identical, at least in regions known to be involved in thedesired regulation. Most preferably, no more than five bases aredifferent.

For the purposes of the appended claims, the term "chimeric protein"refers to a protein which is not identical in sequence to either of twopatental proteins A and B, but which, when its sequence is aligned withthe sequences of A and B, can be seen to borrow features (identically orconservatively) from both parental proteins.

The term "autocrine cell", as used herein, refers to a cell whichproduces a substance which can stimulate the pheromone system pathway ofthat cell. Wild-type α and a cells are not autocrine. However a yeastcell which produces both α-factor and α-factor receptor, or botha-factor and a-factor receptor, in functional form, is autocrine. Byextension, yeast cells which produce a peptide which is being screenedfor the ability to activate the pheromone system pathway (e.g., byactivating a G protein-coupled receptor) are called "autocrine cells",though it might be more precise to call them "putative autocrine cells".Of course, in a library of such cells, in which a multitude of differentpeptides are produced, it is likely that one or more of the cells willbe "autocrine" in the stricter sense of the term.

Farnesyltransferases

The activity of yeast a-factor requires its farnesylation (mediated byprotein farnesyltransferase, comprised of Ram1p and Ram2p), proteolyticcleavage of the C-terminal 3 amino acids of the primary translationproduct (mediated by an as yet unidentified enzyme), andcarboxymethylation of the C-terminal cysteine (mediated by Ste14p). Theyeast and mammalian farnesyltransferases are structurally andfunctionally similar (Gomez R. et al., Biochem. J. 289:25-31, 1993; KohlN. E. et al., J. Biol. Chem. 266:18884-8, 1991). Sequence homologiesexist between the genes encoding the α and β subunits of the yeastfarnesyltransferase (RAM2 and RAM1, respectively) and the genes encodingthe α and β subunits of the mammalian farnesytransferase (Kohl N. E. etal., J. Biol. Chem. 266:18884-8, 1991; Chen W. J. et al., Cell66:327-34, 1991). It has been observed that the β subunit of mammalianfarnesytransferase and Ram1p are 37% identical in amino acid sequence(Chen W. J. et al., Cell 66:327-34, 1991).

The importance of a screen for inhibitors of farnesyltransferase issuggested by the facts that mammalian p21ras, a preeminent regulator ofthe growth and differentiation of mammalian cells that is involved in avariety of cancers, is a substrate for the farnesyltransferase and thatfarnesylation of p21ras is required for its activity. In fact, asynthetic organic inhibitor of farnesyl protein transferase has beenshown to selectively inhibit ras-dependent cell transformation (Kohl etal., Science 260, 1934 (1993). Of the two subunits offarnesyltransferase, the β subunit is a more attractive target forinhibitors, since it is apparently dedicated to farnesylation. The αsubunit, in contrast, is shared by geranylgeranyltransferase I, anenzyme involved in the modification of the Gγ subunits of heterotrimericG proteins and small molecular weight G proteins of the Rho/Rac family.While the β subunit is dedicated to farnesylation, the mammalianfarnesyltransferase has a variety of substrates in addition to p21ras.The effect of inhibitors of the β subunit on the farnesylation of theseother substrates, e.g., lamin proteins, transducin-γ and rhodopsinkinase, will be considered in the design and use of potentialfarnesyltransferase inhibitors.

It has not yet been demonstrated that the homologous mammalian gene willfunctionally substitute for yeast Ram1p, however, this can be formallytested using ram1 mutants and a vector expressing the mammalian geneencoding the β subunit of the farnesyltransferase. If the mammalian βsubunit can function in place of Ram1p, test cells will be both viable(as a result of farnesylation of Ras) and competent for mating (as aresult of farnesylation of a-factor).

If the mammalian gene encoding the β subunit of farnesyltransferasecomplements ram1, yeast would provide a test system for the discovery ofpotential inhibitors of mammalian farnesyltransferase. Specifically,MATa yeast tester cells could be exploited that: 1. carry the gene forthe β subunit of mammalian farnesyltransferase in lieu of RAM1; 2. carrythe cam mutation that renders the strains resistant to loss of Rasfunction in the presence of cAMP; 3. respond to a-factor which theyexport by virtue of heterologous expression of Ste3p; 4. respond toautocrine a-factor such that they cannot grow on media containinggalactose. The latter characteristic will require expression of GAL1under the control of a pheromone-responsive promoter and cellsengineered to contain mutated GAL7 or GAL10 genes. Expression of GAL1 istoxic in the presence of galactose in strains which contain mutations ineither the GAL7 or GAL10 genes. Signaling through the pheromone responsepathway would render cells so engineered galactose-sensitive. Exposureof such strains to compounds which inhibit the β subunit offarnesyltransferase will confer upon these cells the ability to grow onmedia containing galactose and cAMP.

If the mammalian gene encoding the β subunit of farnesyltransferase (andall modified versions of the gene) fails to complement ram1, we may usethe wild-type Ram1p as a surrogate target for potential effectors ofmammalian farnesyltransferase. Specifically, we will use as tester cellsMATa yeast strains that: 1. carry the cam mutation that renders thestrains resistent to loss of RAS function in the presence of cAMP; 2.respond to a-factor which they export by virtue of heterologousexpression of Ste3p; 3. respond to autocrine a-factor such that theycannot grow on media containing galactose. Exposure of such strains tocompounds which inhibit the β subunit of farnesytransferase will conferupon these cells the ability to grow on media containing galactose andcAMP.

In the strategies outlined above, it is desirable to discriminateinhibitors of farnesytransferase from compounds that either directlyblock the negative response to a-factor, e.g. by interfering with theinteraction of the Ste4-Ste18 complex with its effector, or by blockingthe production of a-factor by a mechanism that does not involvefarnesyltransferase. Controls would identify such false positives.Candidate agents will be tested on a MATa strain that is engineered tosecrete α-factor and to respond to the secreted a-factor by failing togrow on galactose-containing media, as in the negative selection schemeoutlined above. The strain will express wild type Ram1p. Any agent thatenables these cells to grow on media containing galactose and cAMP willnot be acting as an inhibitor of farnesyltransferase.

Candidate compounds which pass the foregoing test may act by targetingSte14p, Ste6p, or other proteins involved in the maturation and exportof a-factor, rather than farnesyltransferase. (Note, however, thatcompounds that inhibit processes critical to cell survival will not giverise to false positives. For example, since the protease responsible forthe endoproteolytic removal of the C-terminal tripeptide of the a-factorprecursor likely participates in the processing of Gg and members of theRho/Rac family of proteins, inhibitors of this enzyme may not permitgrowth of the tester cells). Of the proteins involved in the productionof a-factor, only the farnesyltransferase is also a major determinant ofRAS function. Due to this effect, ram1 mutants are defective for growthat 30° C. and completely unable to grow at 37 (He B et al., Proc NatlAcad Sci 88:11373-7, 1991) Tester cells (described above) can be grownin the presence of a candidate inhibitor on galactose-containingmedia±cAMP. If the test compound inhibits farnesyltransferase, cellswill be capable of growth on galactose+cAMP but not on galactose in theabsence of cAMP. This difference may be most obvious at 37°. If, on theother hand, the test compound inhibits other proteins involved ina-factor production, cells will grow on galactose-containing mediaregardless of the presence or absence of cAMP.

Compounds which pass the above tests are likely inhibitors offarnesyltransferase. This can be confirmed and their potenciesdetermined with direct in vitro enzyme assays. Note that the strategiesoutlined will identify farnesyltransferase inhibitors which affectRam1p. Agents which block Ram2p would likely fail to grow under allconditions. Indeed, ram2 null mutations are lethal (He B. et al., ProcNatl Acad Sci 88:11373-7, 1991), perhaps due to the fact that Ram2p alsofunctions as a component of geranylgeranyltransferase I.

Carboxymethyltransferases

In yeast, methylation of the C-terminal amino acid of a-factor, Rasproteins, and presumably Rho/Rac proteins is catalyzed by Ste14p.Although MATa ste14 mutants are unable to mate, reflecting therequirement of carboxymethylation for the activity of a-factor, ste14disruptions are not lethal and do not affect the rate of cellproliferation. Carboxymethylation appears to be dispensible for thefunction of Ras proteins and Ste18p (the yeast homologue of the Gγsubunit). Although Ras function in yeast can apparently tolerate theabsence of carboxymethyl modification, it is nonetheless possible thatinhibitors of mammalian methyltransferases could alter the activity ofmammalian p21ras.

It could be determined if yeast ste14 mutations can be complemented bythe homologous mammalian gene, or a modified version of it. One woulduse an episomal vector to express the mammalian gene encoding themethyltransferase in yeast that are genotypically ste14. The strainwould be a modified MATa strain that expresses the a-factor receptor inlieu of the normal a-factor receptor and that contains an integratedfus1-HIS3 construct, so that the a-factor secreted by the cell confersautocrine growth on histidine-deficient media. If the mammalianmethyltransferase can function in place of Ste14p, the tester cells willbe capable of mating. That is, the mammalian methyltransferase willpermit synthesis of active a-factor in ste14 mutants.

If the mammalian gene encoding the methyltransferase will complementste14, tester strains can be constructed to test for potentialinhibitors of mammalian methyltransferase. In one embodiment, testerMATa yeast strains will: 1. carry a mammalian carboxymethyltransferasegene in lieu of STE14; 2. respond to a-factor which they export byvirtue of heterologous expression of Ste3p; 3. respond to autocrinea-factor such that they cannot grow on media containing galactose as inthe negative GAL1 selection scheme outlined above. Exposure of suchstrains to compounds which inhibit the methyltransferase will conferupon these cells the ability to grow on media containing galactose.

It is desirable to discriminate inhibitors of carboxylmethyltransferaseactivity from compounds that either directly block the negative responseto a-factor, e.g. by interfering with the interaction of the Ste4-Ste18complex with its effector, or block the production of a-factor by amechanism that does not involve methyltransferase. The following controlexperiments will identify such false positives. Candidate inhibitorswill be tested on a MATa strain that is engineered to secrete a-factorand to respond to the secreted a-factor by failing to grow ongalactose-containing media. Any agent that enables these cells to growon media containing galactose will be not be acting as an inhibitor ofcarboxymethyltransferase. Candidate compounds which pass the foregoingtest may be targetting the carboxymethyltransferase,farnesyltransferase, Ste6p, or other proteins involved in the maturationand export of a-factor. In order to discriminate the target of thecompounds, a combination of in vitro biochemical and in vivo geneticassays can be applied: both the carboxymethyltransferase and thefarnesyltransferase can be assayed in vitro to test the effect of thecandidate agent. Furthermore, if the target is Ste14p its overexpressionon high-copy plasmids should confer resistance to the effect of thecompound in vivo.

Proteases

Mature yeast α-factor is a thirteen amino acid peptide that is derivedfrom a polyprotein precursor in much the same manner as mature mammalianmelanocyte-stimulating hormone (MSH) or calcitonin are derived fromprecursor polyproteins. Two genes in the yeast genome encodeprepro-α-factor, MFα1 and MFα2. MFα1 encodes a precursor polypeptidecontaining four copies of mature α-factor embedded in a polypeptide ofthe following structure: hydrophobic pre-sequence/hydrophilicpro-sequence/α-factor/α-factor/α-factor/α-factor. MFα2 encodes apolyprotein precursor of a similar structure containing only two copiesof mature α-factor.

Pre-pro-α-factor is synthesized in the cytoplasm and is then transportedfrom the cytoplasm to the endoplasmic reticulum and then to the Golgialong the classical secretory pathway of S. cerevisiae. The signalsequence of prepro-α-factor is cleaved during transit into the ER bysignal peptidase and asparagine-linked oligosaccharides are added (inthe ER) and modified (in the Golgi) on the pro-segment of the precursoras it transits the secretory pathway. Once in the Golgi, three distinctproteolytic processing events occur. First, the Kex2 protease cleaves atdibasic residues (-KR-) near the amino terminus of each α-factor repeat.Kex2 is homologous to the subtilisin-like endoproteases PC2 and PC1/PC3involved in prohormone processing in mammalian cells (Smeekens andSteiner 1990; Nakayama et al. 1991). Additional mammalian Kex2-likeprocessing endoproteases include PACE, isolated from a human hepatoma,PC4, expressed in testicular germ cells and PC6, a candidate proteasefor the processing of gastrointestinal peptides (Barr et al. 1991;Nakayama et al. 1992; Nakagawa et al. 1993). It appears that Kex2-likeproteins comprise a large family of tissue-specific endoproteases inmammalian cells.

Once Kex2 has released the immature α-factor peptides, two additionalproteases act to complete processing. Kex1 is a specificcarboxypeptidase that removes the carboxy-terminal-KR remaining aftercleavage by Kex2. Like its mammalian counterparts carboxypeptidases Band E, Kex1 is highly specific for peptide substrates withcarboxy-terminal basic residues. The final proteolytic processing eventthat occurs is the removal of the spacer dipeptides at the aminoterminus of each pro-α-factor peptide. This is accomplished by theproduct of the STE13 gene, dipeptidyl aminopeptidase A. This enzyme is atype IV dipeptidyl aminopeptidase: it is capable of cleaving on thecarboxyl side of either -x-A- or -x-P- sites in vitro.

Other type IV dipeptidyl aminopeptidases are believed to be active inthe processing of a variety of pre-peptides in animal cells (Kreil1990). In addition, functional similarity has been proved between yeastKex1 and Kex2 and their mammalian counter-parts in that both yeastenzymes will proteolytically cleave endogenous precursors when expressedin mammalian cells deficient in the native enzyme (Thomas et al. 1988,1990). It appears likely, then, that mammalian homologs of the yeastproteases Kex1, Kex2 and Ste13p, when expressed in yeast, will functionto process a synthetic α-factor pheromone precursor bearing appropriatecleavage sites. Human proteases that may so function in yeast includePC2 and PC1/PC3 (or other Kex2 homologs), carboxypeptidases B and E(Kex1 homologs) and type IV dipeptidyl aminopeptidases (Ste13phomologs).

Yeast would provide a facile assay system for the discovery ofinhibitors of proteases able to process synthetic α-factor. The yeastcould be engineered to express both the potential inhibitor and theexogenous protease, and, preferably, not the latter's yeast cognate.

Furthermore, this means of exploiting yeast pheromone processing toidentify protease inhibitors can be expanded to encompass any proteasethat can be expressed to function in yeast, provided an appropriatecleavage recognition site is included in a synthetic α-factor precursor.In the latter approach, novel proteolytic activities will be added toyeast; these enzymes will substitute for proteases in the α-factormaturation pathway but will not be "catalytic homologues" of Kex1, Kex2or Ste13p. Production of mature α-factor will become dependent on theactivity of the novel protease through removal of the recognitionsite(s) for a selected yeast enzyme from a synthetic MFα gene andinsertion of the recognition sequence for the novel protease(s).

Enzymes for which inhibitors could be identified via this strategyinclude, by way of example, HIV-1 protease or other virally encodedproteases involved in the maturation of infectious particles; neutrophilelastase believed to be involved in pulmonary disease and inflammatorybowel disease; Factor Xa involved in thrombin processing and clottingdisorders; and CD26, a dipeptidyl peptidase IV and putatively the secondreceptor for HIV-1 on CD4⁺ cells. In addition, metalloproteinases (e.g.collagenase) and serine proteases involved in tissue invasion by tumorcells and in metastasis would be suitable therapeutic targets. Insupport of this, it has been demonstrated that administration oftissue-derived inhibitors of metalloproteinases results in decreasedmetastasis in animal models (Schultz et al. 1988). Collagenases havealso been implicated in the destruction of connective tissue whichaccompanies inflammatory processes like rheumatoid arthritis.

The use of the present invention to screen for modulators of prohormoneconvertase PC1 is described in Example 8.

Exogenous ABC Transporters

The majority of proteins destined for transport to the extracellularenvironment proceed through a secretory pathway that includestranslation initiation in the cytoplasm, transport to the lumen of theendoplasmic reticulum, passage through the Golgi to secretory vesiclesand subsequent exit from cells. Other proteins leave the cell by analternative mechanism, which involves mediation by an "ABC transporter".The ABC transporters form a family of evolutionarily conserved proteins,share a similar overall structure, and function in the transport oflarge and small molecules across cellular membranes (Higgins 1992). Thecharacteristic component of members of this protein family is a highlyconserved sequence that binds ATP (Higgins et al., 1986; Hyde et al.1990); these intrinsic membrane proteins are ATPases, deriving energyfrom the hydrolysis of that nucleotide to effect the transport ofmolecules. This family includes over 50 prokaryotic and eukaryoticproteins: transporters of amino acids, sugars, oligosaccharides, ions,heavy metals, peptides, or other proteins belong to this superfamily.Representative transmembrane transporters are included in Table 1.Typically, ABC transporters use the energy of ATP hydrolysis to pumpsubstrate across a cell membrane against a concentration gradient. Someimport substrate, others export it. See Higgins, Ann. Rev. Cell, Biol.,8:67-113 (1992).

The prototypical structure of an ABC transporter includes fourmembrane-associated domains: two hydrophobic, putative membrane-spanningsequences, each predicted to traverse the membrane six times, and twonucleotide binding domains that couple ATP hydrolysis to transport. Inprokaryotes, the domains of an ABC transporter are often present onseparate polypeptides. Various permutations of domain fusions have beendescribed: the E. coli iron hydroxamate transporter contains the twomembrane-spanning domains in a single polypeptide and the ribosetransporter of the same organism bears two nucleotide-binding domains onone molecule. The major histocompatibility complex (MHC) peptidetransporter is composed of two polypeptides, Tap1 and Tap2. TheN-terminus of each protein contains a hydrophobic membrane-spanningdomain while the C-terminus contains an ATP-binding sequence. TogetherTap1 and Tap2 form a functional complex. The heavy metal toleranceprotein, HMT1, expressed in the fission yeast Schizosaccharomyces pombe,consists of a polypeptide containing a single hydrophobic domain and aC-terminal ATP-binding sequence (Ortiz et al. 1992). It may be that theHMT1 transporter functions as a homodimer. The Saccharomyces cerevisiaeSte6 a-factor transporter is expressed as a single polypeptidecontaining two membrane-spanning domains and two nucleotide-bindingdomains. When Ste6 is expressed as two half-molecules, the proteincomplex which apparently forms retains function at a level greater than50% that of the wild type, single polypeptide (Berkower and Michaels1991). In other eukaryotic ABC transporters, including Mdr1, CFTR andMRP, the four domains are also contained within a single polypeptide.Thus, the ABC transporter may be a single multidomain polypeptide, or itmay comprise two or more polypeptides, each providing one or moredomains.

In general, transporters contain six transmembrane segments per eachhydrophobic domain, for a total of twelve segments. The minimum numberof transmembrane segments required for formation of a translocationcomplex appears to be 10. Thus the histidine transporter of S.typhimurium lacks an N-terminal transmembrane segment from each of itshydrophophic domains and therefore contains five transmembrane segmentsper domain (Higgins et al., Nature 298, 723-727 (1982). The MalF proteinof the E. coli maltose transporter contains an N-terminal extension ofhydrophobic sequence which bears two additional transmembrane segments,bringing the total for this hydrophobic domain to 8 (Overduin et al.1988). The N-terminal extension can be deleted, however, without loss offunction of this transporter (Ehrmann et al. 1990). Although the numberof segments required for formation of a functional translocator issuggested by these studies, there exists no data on the precisestructure of the transmembrane segments themselves. These sequences areassumed to have an α-helical form, but this has not been proven and thestructure of the entire translocation complex within the plasma membraneremains to be elucidated.

In order to span the lipid bilayer, a minimum of 20 amino acids isrequired and sequences believed to form transmembrane segments have beenidentified using hydrophobicity scales. Hydrophobicity scales assignvalues to individual amino acid residues indicating the degree ofhydrophobicity of each molecule (Kyte and Doolittle 1982; Engleman etal. 1986). These values are based on experimental data (solubilitymeasurements of amino acids in various solvents, analysis of side chainswithin soluble proteins) and theoretical considerations, and allowprediction of secondary structure in novel sequence with reasonableaccuracy. Analysis using hydrophobicity measurements indicates thosestretches of a protein sequence which have the hydrophobic propertiesconsistent with a transmembrane helix.

With a few exceptions, there is little or no significant amino acidsequence similarity between the transmembrane domains of two differenttransporters. This lack of sequence similarity is not inconsistent withthe apparent function of these hydrophobic domains While these residuesmust be capable of forming the hydrophobic α-helical structures believedto transverse the plasma membrane, many amino acid residues arehydrophobic and can contribute to the formation of an α-helix.

Considerable, if as yet inexplicable, sequence similarity has beendetected in comparisons of the transmembrane domains of the yeast STE6,human MDR and E. coli HlyB hemolysin transporters [Gros et al., Cell 47,371 (1986); McGrath and Varchavsky, Nature 340, 400 (1989); Kuchler etal., EMBO J. 8, 3973 (1989)]. Other sequence similarities can beexplained by gene duplication, as in the case of the transmembranedomains of rodent P-glyco-proteins (Endicott et al. 1991). Thetransmembrane domain of the histidine transporter of S. typhimuriumbears homology to that of the octopine uptake system of Agrobacteriumtumefaciens, the latter two transporters translocate chemically similarsubstrates (Valdiva et al. 1991).

Study of mutant transport proteins has pointed to a role for thetransmembrane sequences in the recognition of substrate. Thus maltosetransporters in E. coli which gain the ability to translocatep-nitrophenyl-α-maltoside bear mutations in the transmembrane domain(Reyes et al. 1986). A mutation in transmembrane segment 11 of MDR hasbeen shown to change the substrate specificity of that transporter (Groset al. 1991) and mutation of charged residues in the transmembranedomain of CFTR changes its ion selectivity (Anderson et al. 1991).

Some aspects of the involvement of extramembrane loop sequences intransport function are being elucidated. In a number of bacterialtransporters a short conserved motif is present on the cytoplasmic loopwhich connects transmembrane segments 4 and 5 [Dassa and Hofnung(1985)]. It has been hypothesized that this sequence interacts with theATP-binding domains of these transport proteins; mutation of thisconserved sequence will abolish transport function (Dassa 1990).Cytoplasmic loops may also be involved in substrate recognition. Thusthe sequences following transmembrane segments 7 and 12 of the yeasta-factor transporter resemble sequences in the a-factor receptor, Ste3p,and may interact with the pheromone substrate (Kuchler et al. 1989). Infact, mutations in the cytoplasmic loops are known to alter thesubstrate specificity of a given transporter. The G185V mutation ofhuman MDR, located in the loop between transmembrane segments 2 and 3,alters the interaction of that transporter with vinblastine andcolchicine (Choi et al. 1988).

The ATP-binding domains are about 200 amino acids long, and domains fromdifferent transporters typically have a sequence identity of 30-50%. Theconserved sequences include the "Walker motifs" which are associatedwith many nucleotide binding proteins. Walker, et al., EMBO J. 1:945-951(1982). Sequence conservation extends over the length of the ATP-bindingdomain, not being limited to the Walker motifs. Furthermore, theATP-binding domains of a single transporter exhibit greater sequenceidentity to one another than to the domains from two differenttransporters. Not all proteins containing a conserved ATP-binding domainare involved in transport, however. The cytoplasmic enzyme UvrAfunctions in DNA repair and the EF-3 protein of yeast is an elongationfactor. Yet both proteins contain ATP-binding cassettes identifiable bysequence comparison.

ATP-binding domains are highly hydrophilic and, in the case oftransporters, appear to reside at the cytoplasmic face of the membrane,anchored there via an association with the membrane-spanning domain ofthese proteins. The points of interaction between the transmembrane andATP-binding domains have not been experimentally determined. Models ofthe structure of the nucleotide binding domain indicate that loopsequences may extend from the core of the structure to interface withthe hydrophilic sequences which transverse the membrane (Hyde et al.1990; Mimura et al. 1991). The two structural models, one based onadenylate cyclase and the other on ras p21 structure, predict a corenucleotide binding fold composed of five β-sheets with the Walker Amotif (a glycine-rich loop) positioned to interact with ATP duringhydrolysis. In addition, loop structures (two loops in one model, onelarge loop in the other) are predicted to extend from the core to couplethe ATP-binding domain to other domains of the transporter. The couplingsequences transmit, most likely through conformational changes theenergy of ATP hydrolysis to those portions of the molecule which areinvolved in transport.

Ste6 function is required for mating but the protein is not necessaryfor yeast survival (Wilson and Herskowiz 1984; Kuchler et al. 1989;McGrath and Varshavsky 1989). Ste6 is structurally homologous to themammalian MDRs. Furthermore, it has been demonstrated that two mammalianMDR proteins, murine Mdr3 and human Mdr1, will substitute functionallyfor the yeast transporter in cells deleted for STE6 (Raymond et al.1992; Kuchler and Thorner 1992). Yeast strains deleted for STE6 serve asa starting point for the design of screens to discover compounds thatmodulate the function of exogenous ABC transporters.

Two different yeast screens can be used to identify modulators of ABCtransporter function. In the first instance, a mammalian protein thattransports a-factor will serve as a target for potential inhibitors oftransporter function. Thus, a yeast strain will be engineered to expressa functional transporter, e.g. mammalian MDR1, which substitutes for theyeast Ste6 protein in the transport of a-factor. Furthermore, thisstrain will be engineered to respond in autocrine fashion to a-factor:e.g., so that the cells will be unable to grow on media containinggalactose. This negative selection will depend on the expression of theGAL1 gene under the control of a pheromone-responsive promoter in astrain background which includes mutated versions of the GAL7 or GAL10genes. Expression of GAL1 in the presence of galactose in such a strainbackground is toxic to cells. In the absence of a-factor transport,signaling down the pheromone response pathway would cease as would theconsequent expression of the toxic gene. Cell growth in the presence ofa test compound, or upon expression of a specific random peptide, wouldsignal inhibition of transport function and the identification of apotential therapeutic.

In addition to inhibitors of MDR, compounds may be identified whichinterfere with the interaction of a-factor with the a-factor receptor.Such compounds can be discriminated by their inhibition ofa-factor-induced growth arrest in a wild type Matα strain. Compounds mayalso impact at other points along the pheromone response pathway toinhibit signaling and these compounds will prevent signal transductionin a wild type Matα strain.

In a second screen, a mutant heterologous transporter (e.g., mutantCFTR) that is initially incapable of transporting a-factor or ana-factor-like peptide can be expressed in autocrine yeast deleted forendogenous Ste6. The cells will be capable of an autocrine response tothe a-factor which those cells produce. Thus a pheromone-responsivepromoter will control expression of a gene that confers an ability togrow in selective media. Such cells will permit identification ofcompounds which correct defects in the transporter and permit it tofunction in the export of pheromone analogues to the extracellularspace. In this way, therapeutic peptides or other classes of chemicalcompounds could be identified which stabilize a mutant protein and allownormal processing, transport, localization to the plasma membrane andfunction. This strategy, if successful, may eliminate the need to"replace" some mutant genes with normal sequence, as envisioned in genetherapies, by recovering the function of mutant proteins through thecorrection of processing and/or localization defects.

In addition to "activators" of the mutant transporter, compounds mayalso be identified which are capable of initiating signalling from thea-factor receptor in the absence of transport by the endogenouslyexpressed pheromone. These compounds will be distinguished by theirability to cause growth arrest in a wild type Matα strain. Compounds mayalso impact at other points along the pheromone pathway and can bediscerned via an ability to initiate signalling in a wild type Matαstrain in the absence of a-factor.

In a preferred embodiment, the exogenous protein produced by the yeastcells is one of the exogenous ABC transporters listed in Table 1.

Exogenous G protein coupled receptors

In some instances, for a drug to cure a disease or alleviate itssymptoms, the drug must be delivered to the appropriate cells, andtrigger the proper "switches." The cellular switches are known as"receptors." Hormones, growth factors, neurotransmitters and many otherbiomolecules normally act through interaction with specific cellularreceptors. Drugs may activate or block particular receptors to achieve adesired pharmaceutical effect. Cell surface receptors mediate thetransduction of an "external" signal (the binding of a ligand to thereceptor) into an "internal" signal (the modulation of a cytoplasmicmetabolic pathway).

In many cases, transduction is accomplished by the following signalingcascade:

An agonist (the ligand) binds to a specific protein (the receptor) onthe cell surface.

As a result of the ligand binding, the receptor undergoes an allostericchange which activates a transducing protein in the cell membrane.

The transducing protein activates, within the cell, production ofso-called "second messenger molecules."

The second messenger molecules activate certain regulatory proteinswithin the cell that have the potential to "switch on" or "off" specificgenes or alter some metabolic process.

This series of events is coupled in a specific fashion for each possiblecellular response. The response to a specific ligand may depend uponwhich receptor a cell expresses. For instance, the response to adrenalinin cells expressing α-adrenergic receptors may be the opposite of theresponse in cells expressing β-adrenergic receptors.

The above "cascade" is idealized, and variations on this theme occur.For example, a receptor may act as its own transducing protein, or atransducing protein may act directly on an intracellular target withoutmediation by a "second messenger".

One family of signal transduction cascades found in eukaryotic cellsutilizes heterotrimeric "G proteins." Many different G proteins areknown to interact with receptors. G protein signaling systems includethree components: the receptor itself, a GTP-binding protein (Gprotein), and an intracellular target protein.

The cell membrane acts as a switchboard. Messages arriving throughdifferent receptors can produce a single effect if the receptors act onthe same type of G protein. On the other hand, signals activating asingle receptor can produce more than one effect if the receptor acts ondifferent kinds of G proteins, or if the G proteins can act on differenteffectors.

In their resting state, the G proteins, which consist of alpha (α), beta(β) and gamma (γ) subunits, are complexed with the nucleotide guanosinediphosphate (GDP) and are in contact with receptors. When a hormone orother first messenger binds to receptor, the receptor changesconformation and this alters its interaction with the G protein. Thisspurs the α subunit to release GDP, and the more abundant nucleotideguanosine triphosphate (GTP), replaces it, activating the G protein. TheG protein then dissociates to separate the α subunit from the stillcomplexed beta and gamma subunits. Either the Gα subunit, or the Gβγcomplex, depending on the pathway, interacts with an effector. Theeffector (which is often an enzyme) in turn converts an inactiveprecursor molecule into an active "second messenger," which may diffusethrough the cytoplasm, triggering a metabolic cascade. After a fewseconds, the Gα converts the GTP to GDP, thereby inactivating itself.The inactivated Gα may then reassociate with the Gβγ complex.

Hundreds, if not thousands, of receptors convey messages throughheterotrimeric G proteins, of which at least 17 distinct forms have beenisolated. Although the greatest variability has been seen in the αsubunit, several different β and γ structures have been reported. Thereare, additionally, several different G protein-dependent effectors.

Most G protein-coupled receptors are comprised of a single protein chainthat is threaded through the plasma membrane seven times. Such receptorsare often referred to as seven-transmembrane receptors (STRs). More thana hundred different STRs have been found, including many distinctreceptors that bind the same ligand, and there are likely many more STRsawaiting discovery.

In addition, STRs have been identified for which the natural ligands areunknown; these receptors are termed "orphan" G protein-coupledreceptors. Examples include receptors cloned by Neote et al. Cell 72,415 (1993); Kouba et al. FEBS Lett. 321, 173 (1993); Birkenbach et al.J. Virol. 67, 2209 (1993).

The "exogenous G protein-coupled receptors" of the present invention maybe any G protein-coupled receptor which is exogenous to the wild-typeyeast cell which is to be genetically engineered for the purpose of thepresent invention. This receptor may be a plant or animal cell receptor.Screening for binding to plant cell receptors may be useful in thedevelopment of, e.g., herbicides. In the case of an animal receptor, itmay be of invertebrate or vertebrate origin. If an invertebratereceptor, an insect receptor is preferred, and would facilitatedevelopment of insecticides. The receptor may also be a vertebrate, morepreferably a mammalian, still more preferably a human, receptor. Theexogenous receptor is also preferably a seven transmembrane segmentreceptor.

Suitable receptors include, but are not limited to, dopaminergic,muscarinic cholinergic, α-adrenergic, β-adrenergic, opioid (includingdelta and mu), cannabinoid, serotoninergic, and GABAergic receptors.Other suitable receptors are listed in Table 2. The term "receptor," asused herein, encompasses both naturally occurring and mutant receptors.

Many of these G protein-coupled receptors, like the yeast a- andα-factor receptors, contain seven hydrophobic amino acid-rich regionswhich are assumed to lie within the plasma membrane. Specific human Gprotein-coupled STRs for which genes have been isolated and for whichexpression vectors could be constructed include those listed in Table 2.Thus, the gene would be operably linked to a promoter functional inyeast and to a signal sequence functional in yeast. Suitable promotersinclude Ste2, Ste3 and gal10. Suitable signal sequences include those ofSte2, Ste3 and of other genes which encode proteins secreted by yeastcells. Preferably, the codons of the gene would be optimized forexpression in yeast. See Hoekema et al., Mol. Cell. Biol., 7:2914-24(1987); Sharp, et al., 14:5125-43 (1986).

The homology of STRs is discussed in Dohlman et al., Ann. Rev. Biochem.,60:653-88 (1991). When STRs are compared, a distinct spatial pattern ofhomology is discernable. The transmembrane domains are often the mostsimilar, whereas the N- and C-terminal regions, and the cytoplasmic loopconnecting transmembrane segments V and VI are more divergent.

The functional significance of different STR regions has been studied byintroducing point mutations (both substitutions and deletions) and byconstructing chimeras of different but related STRs. Synthetic peptidescorresponding to individual segments have also been tested for activity.Affinity labeling has been used to identify ligand binding sites.

It is conceivable that a foreign receptor which is expressed in yeastwill functionally integrate into the yeast membrane, and there interactwith the endogenous yeast G protein. More likely, either the receptorwill need to be modified (e.g., by replacing its V-VI loop with that ofthe yeast STE2 or STE3 receptor), or a compatible G protein should beprovided.

If the wild-type exogenous G protein-coupled receptor cannot be madefunctional in yeast, it may be mutated for this purpose. A comparisonwould be made of the amino acid sequences of the exogenous receptor andof the yeast receptors, and regions of high and low homology identified.Trial mutations would then be made to distinguish regions involved inligand or G protein binding, from those necessary for functionalintegration in the membrane. The exogenous receptor would then bemutated in the latter region to more closely resemble the yeastreceptor, until functional integration was achieved. If this wereinsufficient to achieve functionality, mutations would next be made inthe regions involved in G protein binding. Mutations would be made inregions involved in ligand binding only as a last resort, and then aneffort would be made to preserve ligand binding by making conservativesubstitutions whenever possible.

Preferably, the yeast genome is modified so that it is unable to producethe endogenous a- and α-factor receptors in functional form. Otherwise,a positive assay score might reflect the ability of a peptide toactivate the endogenous G protein-coupled receptor, and not the receptorof interest.

G protein

When the PSP surrogate is an exogenous G protein-coupled receptor, theyeast cell must be able to produce a G protein which is activated by theexogenous receptor, and which can in turn activate the yeasteffector(s). It is possible that the endogenous yeast Gα subunit (e.g.,GPA) will be sufficiently homologous to the "cognate" Gα subunit whichis natively associated with the exogenous receptor for coupling tooccur. More likely, it will be necessary to genetically engineer theyeast cell to produce a foreign Gα subunit which can properly interactwith the exogenous receptor. For example, the Gα subunit of the yeast Gprotein may be replaced by the Gα subunit natively associated with theexogenous receptor.

Dietzel and Kurjan, Cell, 50:1001 (1987) demonstrated that rat Gαsfunctionally coupled to the yeast Gβγ complex. However, rat Gαi2complemented only when substantially overexpressed, while Gα0 did notcomplement at all. Kang, et al., Mol. Cell. Biol., 10:2582 (1990).Consequently, with some foreign Gα subunits, it is not feasible tosimply replace the yeast Gα.

If the exogenous G protein coupled receptor is not adequately coupled toyeast Gβγ by the Gα subunit natively associated with the receptor, theGα subunit may be modified to improve coupling. These modificationsoften will take the form of mutations which increase the resemblance ofthe Gα subunit to the yeast Gα while decreasing its resemblance to thereceptor-associated Gα. For example, a residue may be changed so as tobecome identical to the corresponding yeast Gα residue, or to at leastbelong to the same exchange group of that residue. After modification,the modified Gα subunit might or might not be "substantially homologous"to the foreign and/or the yeast Gα subunit.

The modifications are preferably concentrated in regions of the Gα whichare likely to be involved in Gβγ binding. In some embodiments, themodifications will take the form of replacing one or more segments ofthe receptor-associated Gα with the corresponding yeast Gα segment(s),thereby forming a chimeric Gα subunit. (For the purpose of the appendedclaims, the term "segment" refers to three or more consecutive aminoacids.) In other embodiments, point mutations may be sufficient.

This chimeric Gα subunit will interact with the exogenous receptor andthe yeast Gβγ complex, thereby permitting signal transduction. While useof the endogenous yeast Gβγ is preferred, if a foreign or chimeric Gβγis capable of transducing the signal to the yeast effector, it may beused instead.

Gα Structure

We will now review information regarding Gα structure which is relevantto the design of modified Gα subunits.

In Table 5, part A, the amino terminal 66 residues of GPA1 are alignedwith the cognate domains of human Gαs, Gαi2, Gαi3 and Gα16. In part B,we present alignment of the amino-terminal 66 residues of GPA1₄₁₋ Gαchimeras. In the GPA₄₁₋ Gα hybrids, the amino terminal 41 residues(derived from GPA1) are identical, end with the sequence -LEKQRDKNE- andare underlined for emphasis. All residues following the glutamate (E)residue at position 41 are contributed by the human Gα subunits,including the consensus nucleotide binding motif -GxGxxG-. Periods inthe sequences indicate gaps that have been introduced to maximizealignments in this region. Codon bias is mammalian. For alignments ofthe entire coding regions of GPA1 with Gαs, Gαi, and GαO, Gαq and Gαz,see Dietzel and Kurjan (1987) and Lambright, et al. (1994). Additionalsequence information is provided by Mattera, et al. (1986), Bray, et al.(1986) and Bray, et al. (1987).

The sequences are identified as follows: GPA1 (SEQ ID NO: 82); Gαs (SEQID NO:83); Gαi2 (SEQ ID NO:84); Gαi3 (SEQ ID NO:85); Gα16 (SEQ IDNO:86); GPA41-Gαs (SEQ ID NO:87); GPA41-Gαi2 (SEQ ID NO:88); GPA41-Gαi3(SEQ ID NO:89); and GPA41-Gα16 (SEQ ID NO:90).

The gene encoding a G protein homolog of S. cerevisiae was clonedindependently by Dietzel and Kurjan (1987) (SCG1) and by Nakafuku, etal. (1987) (GPA1). Sequence analysis revealed a high degree of homologybetween the protein encoded by this gene and mammalian Gα. GPA1 encodesa protein of 472 amino acids, as compared with approximately 340-350a.a. for most mammalian Gα subunits in four described families, Gαs,Gαi, Gαq and Gα12/13. Nevertheless, GPA1 shares overall sequence andstructural homology with all Gα proteins identified to date. The highestoverall homology in GPA1 is to the Gαi family (48% identity, or 65% withconservative substitutions) and the lowest is to Gαs (33% identity, or51% with conservative substitutions) (Nakafuku, et al. 1987).

The regions of high sequence homology among Gα subunits are dispersedthroughout their primary sequences, with the regions sharing the highestdegree of homology mapping to sequence that comprises the guaninenucleotide binding/GTPase domain. This domain is structurally similar tothe αβ fold of ras proteins and the protein systhesis elongation factorEF-Tu. This highly conserved guanine nucleotide-binding domain consistsof a six-stranded β sheet surrounded by a set of five α-helices. It iswithin these β sheets and α helices that the highest degree ofconservation is observed among all Gα proteins, including GPA1. Theleast sequence and structural homology is found in the intervening loopsbetween the β sheets and α helices that define the core GTPase domain.There are a total of four "intervening loops" or "inserts" present inall Gα subunits. In the crystal structures reported to date for the GDP-and GTPγS-liganded forms of bovine rod transducin (Noel, et al. 1993;Lambright, et al. 1994), the loop residues are found to be outside thecore GTPase structure. Functional roles for these loop structures havebeen established in only a few instances. A direct role in coupling tophosphodiesterase-γ has been demonstrated for residues within inserts 3and 4 of Gαt (Rarick, et al. 1992; Artemyev, et al. 1992), while a"GAP-like" activity has been ascribed to the largely α-helical insert 1domain of GαS (Markby, et al. 1993).

While the amino- and carboxy-termini of Gα subunits do not sharestriking homology either at the primary, secondary, or tertiary levels,there are several generalizations that can be made about them. First,the amino termini of Gα subunits have been implicated in the associationof Gα with Gβγ complexes and in membrane association via N-terminalmyristoylation. In addition, the carboxy-termini have been implicated inthe association of Gαβγ heterotrimeric complexes with G protein-coupledreceptors (Sullivan, et al. 1987; West, et al. 1985; Conklin, et al.1993). Data in support of these generalizations about the function ofthe N-terminus derive from several sources, including both biochemicaland genetic studies.

As indicated above, there is little if any sequence homology sharedamong the amino termini of Gα subunits. The amino terminal domains of Gαsubunits that precede the first β-sheet (containing the sequence motif-LLLLGAGESG-; see Noel, et al. (1993) for the numbering of thestructural elements of Gα subunits) vary in length from 41 amino acids(GPA1) to 31 amino acids (Gαt). Most Gα subunits share the consensussequence for the addition of myristic acid at their amino termini(MGxxxS-), although not all Gα subunits that contain this motif havemyristic acid covalently associated with the glycine at position 2(speigel, et al. 1991). The role of this post-translational modificationhas been inferred from studies in which the activity of mutant Gαsubunits from which the consensus sequence for myristoylation has beenadded or deleted has been assayed (Mumby, et al. 1990; Linder, et al.1991; Gallego, et al. 1992). These studies suggest two roles forN-terminal myristoylation. First, the presence of amino-terminalmyristic acid has in some cases been shown to be required forassociation of Gα subunits with the membrane, and second, thismodification has been demonstrated to play a role in modulating theassociation of Gα subunits with Gβγ complexes. The role ofmyristoylation of the GPA1 gene product is, at present, unknown.

In other biochemical studies aimed at examining the role of theamino-terminus of Gα in driving the association between Gα and Gβγsubunits, proteolytically or genetically truncated versions of Gαsubunits were assayed for their ability to associate with Gβγ complexes,bind guanine nucleotides and/or to activate effector molecules. In allcases, Gα subunits with truncated amino termini were deficient in allthree functions (Graf, et al. 1992; Journot, et al. 1990; and Neer, etal. 1988). Slepak, et al. (1993) reported a mutational analysis of theN-terminal 56 a.a. of mammalian G≢o expressed in Escherichia coli.Molecules with an apparent reduced ability to interact with exogenouslyadded mammalian Gβγ were identified in the mutant library. As theauthors pointed out, howeve, the assay used to screen the mutants--theextent of ADP-ribosylation of the mutant Gα by pertussis toxin--was nota completely satisfactory probe of interactions between Gα and Gβγ.Mutations identified as inhibiting the interaction of the subunits,using this assay, may still permit the complexing of Gα and Gβγ whilesterically hindering the ribosylation of Gα by toxin.

Genetic studies examined the role of amino-terminal determinants of Gαin heterotrimer subunit association have been carried out in both yeastsystems using GPA1-mammalian Gα hybrids (Kang, et al. 1990) and inmammalian systems using Gαi/Gαs hybrids (Russell and Johnson 1993). Inthe former studies, gene fusions, composed of yeast GPA1 and mammalianGα sequences were constructed by Kang, et al. (1990) and assayed fortheir ability to complement a gpal null phenotype (i.e., constitutiveactivation of the pheromone response pathway) in S. cerevisiae. Kang, etal. demonstrated that wild type mammalian Gαs, Gαi but not Gαo proteinsare competent to associate with yeast Gβγ and suppress the gpa1 nullphenotype, but only when overexpressed. Fusion proteins containing theamino-terminal 330 residues of GPA1 sequence linked to 160, 143, or 142residues of the mammalian Gαs, Gαi and Gαo carboxyl-terminal regions,respectively, also coupled to the yeast mating response pathway whenoverexpressed on high copy plasmids with strong inducible (CUP) orconstitutive (PGK) promoters. All three of these hybrid molecules wereable to complement the gpal null mutation in a growth arrest assay, andwere additionally able to inhibit α-factor responsiveness and mating intester strains. These last two observations argue that hybridyeast-mammalian Gα subunits are capable of interacting directly withyeast Gβγ, thereby disrupting the normal function of the yeastheterotrimer. Fusions containing the amino terminal domain of Gαs, Gαior Gαo, however, did not complement the gpa1 null phenotype, indicatinga requirement for determinants in the amino terminal 330 amino acidresidues of GPA1 for association and sequestration of yeast Gβγcomplexes. Taken together, these data suggest that determinants in theamino terminal region of Gα subunits determine not only the ability toassociate with Gβγ subunits in general, but also with specific Gβγsubunits in a species-restricted manner.

Hybrid Gαi/Gαs subunits have been assayed in mammalian expressionsystems (Russell and Johnson 1993). In these studies, a large number ofchimeric Gα subunits were assayed for an ability to activate adenylylcyclase, and therefore, indirectly, for an ability to interact with Gβγ(i.e., coupling of Gα to Gβγ=inactive cyclase; uncoupling of Gα fromGβγ=active cyclase). From these studies a complex picture emerged inwhich determinants in the region between residues 25 and 96 of thehybrids were found to determine the state of activation of these allelesas reflected in their rates of guanine nucleotide exchange and GTPhydrolysis and the extent to which they activated adenylyl cyclase invivo. These data could be interpreted to support the hypothesis thatstructural elements in the region between the amino terminal methionineand the β1 sheet identified in the crystal structure of Gαt (see Noel,et al. 1993 and Lambright, et al. 1994) are involved in determining thestate of activity of the heterotrimer by (1) drivingassociation/dissociation between Gα and Gβγ subunits; (2) drivingGDP/GTP exchange. While there is no direct evidence provided by thesestudies to support the idea that residues in this region of Gα andresidues in Gβγ subunits contact one another, the data nonethelessprovide a positive indication for the construction of hybrid Gα subunitsthat retain function. There is, however, a negative indicator thatderives from this work in that some hybrid constructs resulted inconstitutive activation of the chimeric proteins (i.e., a loss ofreceptor-dependent stimulation of Gαβγ dissociation and effectoractivation).

Construction of chimeric Gα subunits

In designing Gα subunits capable of transmitting, in yeast, signalsoriginating at mammalian G protein-coupled receptors, two generaldesiderata were recognized. First, the subunits should retain as much ofthe sequence of the native mammalian proteins as possible. Second, thelevel of expression for the heterologous components should approach, asclosely as possible, the level of their endogenous counterparts. Theresults described by King, et al. (1990) for expression of the humanβ2-adrenergic receptor and Gαs in yeast, taken together with negativeresults obtained by Kang, et al. (1990) with full-length mammalian Gαsubunits other than Gαs, led us to the following preferences for thedevelopment of yeast strains in which mammalian G protein-coupledreceptors could be linked to the pheromone response pathway

1. Mammalian Gα subunits will be expressed using the native sequence ofeach subunit or, alternatively, as minimal gene fusions with sequencesfrom the amino terminus of GPA1 replacing the homologous residues fromthe mammalian Gα subunits.

2. Mammalian Gα subunits will be expressed from the GPA1 promotor eitheron low copy plasmids or after integration into the yeast genome as asingle copy gene.

3. Endogenous Gβγ subunits will be provided by the yeast STE4 and STE18loci.

Site-Directed Mutagenesis versus Random Mutagenesis

There are two general approaches to solving structure-function problemsof the sort presented by attempts to define the determinants involved inmediating the association of the subunits that comprise the G proteinheterotrimer. The first approach, discussed above with respect to hybridconstructs, is a rational one in which specific mutations or alterationsare introduced into a molecule based upon the available experimentalevidence. In a second approach, random mutagenesis techniques, coupledwith selection or screening systems, are used to introduce large numbersof mutations into a molecule, and that collection of randomly mutatedmolecules is then subjected to a selection for the desired phenotype ora screen in which the desired phenotype can be observed against abackground of undesirable phenotypes. With random mutagenesis one canmutagenize an entire molecule or one can proceed by cassettemutagenesis. In the former instance, the entire coding region of amolecule is mutagenized by one of several methods (chemical, PCR, dopedoligonucleotide synthesis) and that collection of randomly mutatedmolecules is subjected to selection or screening procedures. Randommutagenesis can be applied in this way in cases where the molecule beingstudied is relatively small and there are powerful and stringentselections or screens available to discriminate between the differentclasses of mutant phenotypes that will inevitably arise. In the secondapproach, discrete regions of a protein, corresponding either to definedstructural (i.e. a-helices, b-sheets, turns, surface loops) orfunctional determinants (e.g., catalytic clefts, binding determinants,transmembrane segments) are subjected to saturating or semi-randommutagenesis and these mutagenized cassettes are re-introduced into thecontext of the otherwise wild type allele. Cassette mutagenesis is mostuseful when there is experimental evidence available to suggest aparticular function for a region of a molecule and there is a powerfulselection and/or screening approach available to discriminate betweeninteresting and uninteresting mutants. Cassette mutagenesis is alsouseful when the parent molecule is comparatively large and the desire isto map the functional domains of a molecule by mutagenizing the moleculein a step-wise fashion, i.e. mutating one linear cassette of residues ata time and then assaying for function.

We are applying random mutagenesis in order to further delineate thedeterminants involved in Gα-Gβγ association. Random mutagenesis may beaccomplished by many means, including:

1. PCR mutagenesis, in which the error prone Taq polymerase is exploitedto generate mutant alleles of Gα subunits, which are assayed directly inyeast for an ability to couple to yeast Gβγ.

2. Chemical mutagenesis, in which expression cassettes encoding Gαsubunits are exposed to mutagens and the protein products of the mutantsequences are assayed directly in yeast for an ability to couple toyeast Gβγ.

3. Doped synthesis of oligonucleotides encoding portions of the Gα gene.

4. In vivo mutagenesis, in which random mutations are introducted intothe coding region of Gα subunits by passage through a mutator strain ofE. coli, XL1-Red (mutD5 mutS mutT) (Stratagene, Menasa, Wis.).

The random mutagenesis may be focused on regions suspected to beinvolved in Gα-Gβγ association as discussed in the next section. Randommutagenesis approaches are feasible for two reasons. First, in yeast onehas the ability to construct stringent screens and facile selections(growth vs. death, transcription vs. lack of transcription) that are notreadily available in mammalian systems. Second, when using yeastit ispossible to screen effienctly through thousands of transformantsrapidly.

Cassette mutagenesis is immediately suggested by the observation (seeinfra) that the GPA₄₁ hybrids couple to the pheromone response pathway.This relatively small region of Gα subunits represents a reasonabletarget for this type of mutagenesis. Another region that may be amenableto cassette mutagenesis is that defining the surface of the switchregion of Gα subunits that is solvent-exposed in the crystal structuresof Gαi1 and transducin. From the data described below, this surface maycontain residues that are in direct contact with yeast Gβγ subunits, andmay therefore be a reasonable target for mutagenesis.

Rational Design of Chimeric Gα Subunits

Several classes of rationally designed GPA1-mammalian Gα hybrid subunitshave been tested for the ability to couple to yeast βγ. The first, andlargest, class of hybrids are those that encode different lengths of theGPA1 amino terminal domain in place of the homologous regions of themammalian Gα subunits. This class of hybrid molecules includesGPA_(BamHI), GPA₄₁, GPA_(ID), and GPA_(LW) hybrids, described below. Therationale for constructing these hybrid Gα proteins is based on results,described above, that bear on the importance of the amino terminalresidues of Gα₋₋ in mediating interaction with Gβγ.

Preferably, the yeast Gα subunit is replaced by a chimeric Gα subunit inwhich a portion, e.g., at least about 20, more preferably at least about40, amino acids, which is substantially homologous with thecorresponding residues of the amino terminus of the yeast Gα, is fusedto a sequence substantially homologous with the main body of a mammalian(or other exogenous) Gα. While 40 amino acids is the suggested startingpoint, shorter or longer portions may be tested to determine the minimumlength required for coupling to yeast Gβγ and the maximum lengthcompatible with retention of coupling to the exogenous receptor. It ispresently believed that only the final 10 or 20 amino acids at thecarboxy terminus of the Gα subunit are required for interaction with thereceptor.

GPA_(BamHI) hybrids. Kang et al. (1990) described hybrid Gα₋₋ subunitsencoding the amino terminal 310 residues of GPA1 fused to the carboxylterminal 160, 143 and 142 residues, respectively, of GαS, Gαi2, and Gαo.In all cases examined by Kang et al., the hybrid proteins were able tocomplement the growth arrest phenotype of gpa1 strains. We haveconfirmed these findings and, in addition, have constructed and testedhybrids between GPA1 and Gαi3, Gαq and Gα16 (see Example 11). Allhybrids of this type that have been tested functionally complement thegrowth arrest phenotype of gpa1 strains.

GPA₄₁ hybrids. The rationale for constructing a minimal hybrid encodingonly 41 amino acids of GPA1 relies upon the biochemical evidence for therole of the amino-terminus of Gα subunits discussed above, together withthe following observation. Gβ and Gγ subunits are known to interact viaα-helical domains at their respective amino-termini (Pronin, et al.1992; Garritsen, et al. 1993). The suggestion that the amino termini ofGα subunits may form an helical coil and that this helical coil may beinvolved in association of Gα with Gβγ (Masters, Stroud, and Bourne1986; Lupas, Lupas and Stock 1992) leads to the hypothesis that thethree subunits of the G-protein heterotrimer interact with one anotherreversibly through the winding and unwinding of their amino-terminalhelical regions. A mechanism of this type has been suggested, as well,from an analysis of leucine zipper mutants of the GCN4 transcriptionfactor (Harbury, et al. 1993). The rationale for constructing hybridslike those described by Kang, et al. (1990), that contain a majority ofyeast sequence and only minimal mammalian sequence, derives from theirability to function in assays of coupling between Gα and Gβγ subunitsHowever, these chimeras had never been assayed for an ability to coupleto both mammalian G protein-coupled receptors and yeast Gβγ subunits,and hence to reconstitute a hybrid signalling pathway in yeast.

GPA₄₁ hybrids that have been constructed and tested include Gαs, Gαi2,Gαi3, Gαq, Gαo_(a), Gαo_(b) and Gα16 (see Example 11). Hybrids of Gαs,Gαi2, Gαi3, and Gα16 functionally complement the growth arrest phenotypeof gpa1 strains, while GPA₄₁ hybrids of Gαo_(a) and Gαo_(b) do not. Inaddition to being tested in a growth arrest assay, these constructs havebeen assayed in the more sensitive transcriptional assay for activationof a fus1p-HIS3 gene. In both of these assays, the GPA₄₁ -Gαs hybridcouples less well than the GPA₄₁ -i2, -i3, and -16 hybrids, while theGPA₄₁ -o_(a) and -o_(b) hyrids do not function in either assay.

Several predictive algorithms indicate that the amino terminal domain upto the highly conserved sequence motif -LLLLGAGESG- (the first L in thismotif is residue 43 in GPA1) forms a helical structure with amphipathiccharacter. Assuming that a heptahelical repeat unit, the followinghybrids between GPA1 and GaS can be used to define the number of helicalrepeats in this motif necessary for hybrid function:

GPA1-7/Gαs8-394

GPA1-14/Gαs15-394

GPA1-21/Gαs22-394

GPA1-28/Gαs29-394

GPA1-35/Gαs36-394

GPA1-42/Gαs43-394

In this hybrids, the prediction is that the structural repeat unit inthe amino terminal domain up to the tetra-leucine motif is 7, and thatswapping sequences in units of 7 will in effect amount to a swap of unitturns of turns of the belical structure that comprises this domain.

A second group of "double crossover" hybrids of this class are thosethat are aligned on the first putative heptad repeat beginning withresidue G11 in GPA1. In these hybrids, helical repeats are swapped fromGPA1 into a GaS backbone one heptad repeat unit at a time.

GαS1-10/GPA11-17/Gαs18-394

GαS1-17/GPA18-24/GαS25-394

GαS1-17/GPA25-31/GαS32-394

GαS1-17/GPA32-38/GαS39-394

The gap that is introduced between residues 9 and 10 in the GaS sequenceis to preserve the alignment of the -LLLLGAGE- sequence motif.

This class of hybrids can be complemented by cassette mutagenesis ofeach heptad repeat followed by screening of these collections of"heptad" libraries in standard coupling assays.

A third class of hybrids based on the prediction that the amino terminusforms a helical domain with a heptahelical repeat unit are those thateffect the overall hydrophobic or hydrophilic character of the opposingsides of the predicted helical structure (See Lupas, Stock and Stock).In this model, the α and d positions of the heptad repeat abcdefg arefound to be conserved hydrophobic residues that define one face of thehelix, while the e and g positions define the charged face of the helix.

In this class of hybrids, the sequence of the GaS parent is maintainedexcept for specific substitutions at one or more of the followingcritical residues to render the different helical faces of GaS more"GPA1-like"

K8Q

+I-10

E10G

Q12E

R13S

N14D

E15P

E15F

K17L

E21R

K28Q

K32L

V36R

This collection of single mutations could be screened for couplingeffieciency to yeast Gβγ and then constructed in combinations (doubleand greater if necessary).

A fourth class of hybrid molecules that span this region of GPA1-Gαhybrids are those that have junctions between GPA1 and Gα subunitsintroduced by three primer PCR. In this approach, the two outsideprimers are encoded by sequences at the initiator methionine of GPA1 onthe 5' side and at the tetraleucine motif of GαS (for example) on the 3'side. A series of junctional primers spanning different junctionalpoints can be mixed with the outside primers to make a series ofmolecules each with different amounts of GPA1 and GaS sequences,respectively.

GPA_(ID) and GPA_(LW) hybrids. The regions of high homology among Gαsubunits that have been identified by sequence alignment areinterspersed throughout the molecule. The G1 region containing thehighly conserved -GSGESGDST- motif is followed immediately by a regionof very low sequence consevation, the "i1" or insert 1 region. Bothsequence and length vary considerably among the i1 regions of the Gαsubunits. By aligning the sequences of Gα subunits, the conservedregions bounding the i1 region were identified and two additionalclasses of GPA1-Gα hybrids were constructed. The GPA_(ID) hybrids encodethe amino terminal 102 residues of GPA1 (up to the sequence -QARKLGIQ-)fused in frame to mammalian Gα subunits, while the GPA_(LW) hybridsencode the amino terminal 244 residues of GPA1 (up to the sequence-LIHEDIAKA- in GPA1). The reason for constructing the GPA_(ID) andGPA_(LW) hybrids was to test the hypothesis that the i1 region of GPA1is required for mediating the interaction of GPA1 with yeast Gβγsubunits, for the stable expression of the hybrid molecules, or forfunction of the hybrid molecules. The GPA_(ID) hybrids contain the aminoterminal domain of GPA1 fused to the i1 domain of mammalian subunits,and therefore do not contain the GPA1 i1 region, while the GPA_(LW)hybrids contain the amino terminal 244 residues of GPA1 including theentire i1 region (as defined by sequence alignments). Hybrids of bothGPA_(ID) and GPA_(LW) classes were constructed for GαS, Gαi2, G+i3,Gαo_(a), and Gα16; none of these hybrids complemented the gpa1 growtharrest phenotype.

Subsequent to the construction and testing of the GPA_(ID) and GPA_(LW)classes of hybrids, the crystal structures of Gα_(transducin) in boththe GDP and GTPγS-liganded form, and the crystal structure of severalGαi1 variants in the GTPγS-liganded and GDP-AlF₄ forms were reported(Noel et al. 1993; Lambright et al. 1994 and Coleman et al. 1994). Thecrystal structures reveal that the i1region defined by sequencealignment has a conserved structure that is comprised of six alphahelices in a rigid array, and that the junctions chosen for theconstruction of the GPA_(ID) and GPA_(LW) hybrids were not compatiblewith conservation of the structural features of the i1 region observedin the crystals. The junction chosen for the GPA_(ID) hybrids falls inthe center of the long αA helix; chimerization of this helix in alllikelihood destabilizes it and the protein structure in general. Thesame is true of the junction chosen for the GPA_(LW) hybrids in whichthe crossover point between GPA1 and the mammalian Gα subunit falls atthe end of the short αC helix and therefore may distort it anddestabilize the protein.

The failure of the GPA_(ID) and GPA_(LW) hybrids is predicted to be dueto disruption of critical structural elements in the il region asdiscussed above. Based upon new alignments and the data presented inNoel et al (1993), Lambright et al (1994), and Coleman et al (1994),this problem can be averted with the following hybrids. In thesehybrids, the junctions between the ras-like core domain and the ilhelical domain are introduced outside of known structural elements likealpha-helices.

    ______________________________________                                        Hybrid A  GαS1-67/GPA66-299/GαS203-394                            ______________________________________                                    

This hybrid contains the entire il insert of GPA1 interposed into theGαS sequence.

    ______________________________________                                        Hybrid B GPA1-41/GαS4443-67/GPA66-299/GαS203-394                  ______________________________________                                    

This hybrid contains the amino terminal 41 residues of GPA1 in place ofthe 42 amino terminal residues of GαS found in Hybrid A.

Gαs Hybrids. There is evidence that the "switch region" encoded byresidues 171-237 of Gα transducin (using the numbering of Noel et al(1993)) also plays a role in Gβγ coupling. First, the G226A mutation inGαS (Miller et al. 1988) prevents the GTP-induced conformational changethat occurs with exchange of GDP for GTP upon receptor activation byligand. This residue maps to the highly conserved sequence -DVGGQ-,present in all Gα subunits and is involved in GTP hydrolysis. In boththe Gαt and Gαi1 crystal structures, this sequence motif resides in theloop that connects the ₋₋ β3 sheet and the α2 helix in the guaninenucleotide binding core. In addition to blocking the conformationalchange that occurs upon GTP binding, this mutation also preventsdissociation of GTP-liganded Gαs from Gβγ. Second, crosslinking datareveals that a highly conserved cysteine residue in the α2 helix (C215in Gαo, C210 in Gαt) can be crosslinked to the carboxy terminal regionof Gβ₋₋ subunits. Finally, genetic evidence (Whiteway et al. 1993)identifies an important single residue in GPA1 (E307) in the β2 sheet ofthe core structure that may be in direct contact with βγ. A mutation inthe GPA1 protein at this position suppresses the constitutive signallingphenotype of a variety of STE4 (Gβ) dominant negative mutations that arealso known to be defective in Gα-Gβγ association (as assessed intwo-hybrid assay in yeast as well as by more conventional genetictests).

We have tested the hypothesis that there are switch region determinantsinvolved in the association of Gα with Gβγ by constructing a series ofhybrid Gα proteins encoding portions of GPA1 and GαS in differentcombinations (FIG. 11). The hybrid proteins were tested in bothbiological assay described above and the results are summarized in Table6.

Two conclusions may be drawn. First, in the context of the aminoterminus of GαS, the GPA1 switch region suppresses coupling to yeast Gβγ(SGS), while in the context of the GPA1 amino terminus the GPA1 switchregion stabilizes coupling with Gβγ (GPA₄₁ -SGS). This suggests thatthese two regions of GPA1 collaborate to allow interactions between Gαsubunits and Gβγ subunits. This conclusion is somewhat mitigated by theobservation that the GPA₄₁ -Gαs hybrid that does not contain the GPA1switch region is able to complement the growth arrest phenotype of gpa1strains. We have not to date noted a quantitative difference between thebehaviour of the GPA₄₁ -Gαs allele and the GPA₄₁ -SGS allele, but ifthis interaction is somewhat degenerate, then it may be difficult toquantitate this accurately. The second conclusion that can be drawn fromthese results is that there are other determinants involved instabilizing the interaction of Gα with Gβγ beyond these two regions asnone of the GPA1/Gαs hybrid proteins couple as efficiently to yeast Gβγas does native GPA1.

The role of the surface-exposed residues of this region may be crucialfor effective coupling to yeast Gβγ, and can be incorporated into hybridmolecules as follows below.

    ______________________________________                                        GαS-GPA-Switch                                                                      GαS 1-202/GPA298-350/GαS 253-394                      ______________________________________                                    

This hybrid encodes the entire switch region of GPA1 in the context ofGaS.

    ______________________________________                                        GαS-GPA-α2                                                                   GαS 1-226/GPA322-332/GαS 238-394                       ______________________________________                                    

This hybrid encodes the a² belix of GPA1 in the context of GaS.

    ______________________________________                                        GPA41-GαS-GPA-α2                                                                GPA1--41/GαS43-226/GPA322-332/                                          GαS238-394                                                ______________________________________                                    

This hybrid encodes the 41 residue amino terminal domain of GPA1 and theα2 helix of GPA1 in the context of GαS.

Finally, the last class of hybrids that will be discussed here are thosethat alter the surface exposed residues of the β2 and β3 sheets of αS sothat they resemble those of the GPA1 αs helix. These altered α2 helicaldomains have the following structure. (The positions of the alteredresidues correspond to GαS.)

L203K

K211E

D215G

K216S

D229S

These single mutations can be engineered into a GαS backbone singly andin pairwise combinations. In addition, they can be introduced in thecontext of both the full length GαS and the GPA₄₁ -GαS hybrid describedpreviously. All are predicted to improve the coupling of Gα subunits toyeast Gβγ subunits by virtue of improved electrostatic and hydrophobiccontacts between this region and the regions of Gβ defined by Whitewayand co-workers (Whiteway et al (1994) that define site(s) that interactwith GPA1).

Summary.₋₋ Identification of hybrid Gα subunits that couple to the yeastpheromone pathway has led to the following general observations. First,all GPA_(BamHI) Hybrids associate with yeast Gβγ, therefore at a minimumthese hybrids contain the determinants in GPA1 necessary for coupling tothe pheromone response pathway. Second, the amino terminal 41 residuesof GPA1 contain sufficient determinants to facilitate coupling of Gαhybrids to yeast Gβγ in some, but not all, instances, and that some Gαsubunits contain regions outside of the first 41 residues that aresufficiently similar to those in GPA1 to facilitate interaction withGPA1 even in the absence of the amino terminal 41 residues of GPA1.Third, there are other determinants in the first 310 residues of GPA1that are involved in coupling Gα subunits to yeast Gβγ subunits.

The vavious classes of hybrids noted above are not mutually exclusive.For example, a Gα containing GPA1-41 could also feature the L203Kmutation.

While, for the sake of simplicity, we have described hybrids of yeastGPA1 and a mammalian Gαs, it will be appreciated that hybrids may bemade of other yeast Gα subunits and/or other mammalian Gα subunits,notably mammalian Gαi subunits. Moreover, while the described hybridsare constructed from two parental proteins, hybrids of three or moreparental proteins are also possible.

As shown in the Examples, chimeric Gα subunits have been especiallyuseful in coupling receptors to Gαi species.

Expression of Gα

Kang et al. (1990) reported that several classes of native mammalian Gαsubunits were able to interact functionally with yeast βγ subunits whenexpression of Gα was driven from a constitutively active, strongpromotor (PGK) or from a strong inducible promotor (CUP). These authorsreported that rat GαS, Gαi2 or Gαo expressed at high level coupled toyeast βγ. High level expression of mammalian Gα₋₋ (i.e.non-stoichiometric with respect to yeast βγ) is not desirable for useslike those described in this application. Reconstruction of Gprotein-coupled receptor signal transduction in yeast requires thesignalling component of the heterotrimeric complex (Gβγ) to be presentstoichiometrically with Gα subunits. An excess of Gα subunits (as wasrequired for coupling of mammalian Gαi2 and Gαo to yeast Gβγ₋₋ in Kanget al.) would dampen the signal in systems where Gβγ subunits transducethe signal. An excess of Gβγ subunits raises the background level ofsignalling in the system to unacceptably high levels.

Preferably, levels of Gα and Gβγ subunits are balanced. For example,heterologous Gα subunits may be expressed from a low copy (CEN ARS)vector containing the endogenous yeast GPA1 promotor and the GPA1 3'untranslated region. The minimum criterion, applied to a heterologous Gαsubunit with respect to its ability to couple functionally to the yeastpheromone pathway, is that it complement a gpa1 genotype when expressedfrom the GPA1 promoter on low copy plasmids or from an integrated,single copy gene. In the work described in this application, allheterologous Gα subunits have been assayed in two biological systems. Inthe first assay heterologous Gα subunits are tested for an ability tofunctionally complement the growth arrest phenotype of gpa1 strains. Inthe second assay the transcription of a fus1-HIS3 reporter gene is usedto measure the extent to which the pheromone response pathway isactivated, and hence the extent to which the heterologous Gα subunitsequesters the endogenous yeast Gβγ complex.

Mammalian Gαs, Gαi2, Gαi3, Gαq, Gα11, Gα16, Gαo_(a), Gαo_(b), and Gαzfrom rat, murine or human origins were expressed from a low copy, CENARS vector containing the GPA1 promoter Functional complementation ofgpa1 strains was not observed in either assay system with any of thesefull-length Gα constructs with the exception of rat and human GαS.

Chimeric Yeast βγ subunits

An alternative to the modification of a mammalian Gα subunit forimproved signal transduction is the modification of the pertinent sitesin the yeast Gβ or Gγ subunits. The principles discussed already withrespect to Gα subunits apply, mutatis mutandis, to yeast Gβ or Gγ.

For example, it would not be unreasonable to target the yeast Ste4p Gβsubunit with cassette mutagenesis. Specifically, the region of Ste4pthat encodes several of the dominant negative, signalling-defectivemutations would be an excellent target for cassette mutagenesis whenlooking for coupling of yeast Gβγ to specific mammalian Gα subunits.

Protein kinases

Mitogen-activated protein kinase (MAP kinase) and its activator, MAPkinase kinase or MEK, are believed to be key molecules in thetransduction of intracellular signals in mammalian cells. The activityof MAPK, a serine/threonine protein kinase, has been shown to depend onits phosphorylation by the dually specific MEK at tyrosine and threonineresidues. MEK activity, in turn, depends on its phosphorylation onserine and threonine by a third kinase, MAP kinase kinase kinase, orMEKK, whose function in some systems is fulfilled by the protooncogeneproduct Raf1p.

An essential part of the S. cerevisiae pheromone signalling pathway iscomprised of a protein kinase cascade composed of the products of theSTE11, STE7, and FUS3/KSS1 genes (the latter pair are distinct andfunctionally redundant). Functional studies have established thedependence of FUS3p activity on tyrosine and threonine phosphorylationby STE7p, whose activity is regulated by its phosphorylation by STE11p.A second protein kinase cascade, responsive to protein kinase C, hasbeen identified in S. cerevisiae. When this pathway is disrupted, yeastcells lose their ability to grow in media of low osmotic strength.Although its components have not been characterized to the same extentas that of the mating pathway cascade, sequence analysis identifiesBCK1p as a MEKK, MKK1p/MKK2p as MEKs, and MPK1p as a MAPK.

Kinase signalling pathways appear to be conserved among eukaryotes. Thussignificant sequence homology is found between Xenopus MAP kinase andthe products of the following yeast kinase genes: FUS3, KSS1, MPK1 (S.cerevisiae) and Spk1 (Schizosaccharomyces pombe). In addition, mammalianMEK has been found to be homologous to the products of STE7 (S.cerevisiae) and Byr1 (S. pombe) [Crews et al. Science 258, 478 (1992)].Functional homologies between some kinases has been demonstrated throughsubstitution of heterologous kinase genes in yeast kinase deletionmutants. Thus Xenopus MAP kinase will complement an mpk1Δ mutant in S.cerevisiae (however, this kinase will not substitute for Fus3p or Kss1pfunction in the same organism) [Lee et al. Mol. Cell. Biol. 13, 3067(1993)]. Both mammalian and Xenopus MAP kinase will substitute for Spk1function in S. pombe [Neiman et al. Molec. Biol. Cell. 4, 107 (1993);Gotoh et al. Molec. Cell. Biol. 13, 6427 (1993)]. Rabbit MAP kinasekinase will complement a byr1 defect in S. pombe but only whenco-expressed with Raf1 kinase; the latter thus appears to be a directactivator of MEK (Hughes et al. Nature 364, 349 (1993).

The use of the instant invention to screen for modulators of human MEKis described in Example 9.

Cyclins

Members of another mammalian protein kinase family, one active inprogression through the cell cycle, have been identified bycomplementation of cell cycle kinase mutants in yeast. The humanhomologue of p34cdc2 (S. pombe) and p34cdc28 (S. cerevisiae), proteinswhich control the progression to DNA synthesis and mitosis in yeast, wasidentified by complementation of a cdc2 mutation in S. pombe [Lee andNurse, Nature 327, 31-35 (1987)]. CDK2, a second human p34 homologue,was identified by functional complementation of p34cdc28 mutations in S.cerevisiae [Elledge and Spottswood, EMBO J. 10, 2653 (1991)]. Activationof p34 depends on its association with regulatory subunits, termedcyclins. Tight control of cyclin expression as well as the inherentinstability of these proteins once expressed contribute to a regulatedactivation of p34 kinase and progression through the cell cycle.

A number of putative G1 human cyclins have been identified through theirability to substitute for the yeast G1 cyclins, CLN1, CLN2 and CLN3, inthe pheromone signalling pathway. Thus human cyclins C, D and E wereidentified by their ability to rescue cln⁻ yeast from growth arrest [Lewet al., Cell 66, 1197 (1991)]. It has also been demonstrated that otherclasses of human cyclins can substitute functionally for the CLNproteins in yeast. The human cyclins A, B1 and B2 (cyclins normallyassociated with governance of mitosis) will also function as G1 cyclinsin S. cerevisiae [Lew et al., Cell 66, 1197 (1991)].

Certain cyclins are periodically accumulating regulators of the p34kinase (p34cdc2 in humans and p34cdc28 in S. cerevisiae) . The p34kinase is functionally conserved from yeast to humans and the activityof this threonine/serine-specific protein kinase is required if cellsare to progress through several checkpoints in the cell cycle, includingone that is termed START. START occurs late in the G1 phase and is theswitch point for cells between the quiescent state and proliferation.The kinase is activated at discrete points in the cell cycle through itsassociation with specific cyclins, regulatory subunits which are alsofunctionally conserved among eukaryotes. Three cyclins appear to operatein progression through START in S. cerevisiae, CLN1, CLN2 and CLN3(Hadwiger et al. 1989; Cross 1988; Nash 1988). The sequences of the CLNproteins bear some homology to those of mammalian A- and B-type cyclins;these proteins are believed to regulate S (DNA synthesis) and M(mitotic) phases of the mammalian cell cycle.

Sequence comparisons among the cyclin proteins identified in differentspecies indicates that a region of high sequence conservation iscontained within an approximately 87 residue domain that generallycomprises central cyclin sequence but which is located near the aminoterminus of the yeast G1 cyclins. This conserved domain is termed the"cyclin box". A second region of homology shared by most of the cyclinsis a C-terminal sequence rich in proline, glutamate, serine, threonineand aspartate residues flanked by basic amino acids that is termed aPEST motif (Rogers et al. 1986). PEST motifs are found in unstableproteins and are believed to signal for constitutive ubiquitin-mediateddegradation. The degradation of cyclins A and B is signalled via adifferent sequence, a "mitotic destruction motif" (Glotzer et al. 1991),that is not shared by other mammalian cyclins.

Sequence comparisons made between the yeast CLN proteins and the humanA, B, C, D and E cyclins indicate the existence of appreciablehomologies (Lew et al. 1991). Across the most conserved regions,including the cyclin box, human cyclin C bears 18% sequence identityboth to human cyclins D and E and to the yeast CLN proteins. Humancyclins D and E appear to be more related to human A- and B-type cyclins(33% identical) than to the yeast CLNs (24% identical).

All human cyclins identified to date will substitute functionally foryeast cyclins. In fact, the mammalian cyclins C, D1 and E wereidentified through their ability to complement defective CLN function inyeast (Lew et al. 1991). Mammalian A- and B-type cyclins also substitutefunctionally for the CLN proteins in yeast, therefore this abilitycannot definitively mark a mammalian cyclin as one that would operate inG1. However, the cyclins C, D1 and E have been shown to be expressed inG1 in mammalian cells and the expression pattern of cyclin E during thecell cycle most closely parallels the expression patterns observed forthe yeast G1 cyclins (Lew et al. 1991; Koff et al. 1991).

In mammalian cells, cyclin C mRNA accumulates early in G1 while cyclin Eaccumulates late in that phase. D cyclin mRNA levels are insufficient toallow tracking of expression patterns in human cells and the role ofthis cyclin is therefore not clear (Lew et al. 1991). In mouse cells,the D1 gene, CYL1, is expressed in the G1 phase and the D1 gene appearsto be regulated by colony-stimulating factor 1 (Matsushime et al. 1991).Expression of D1 cyclin is highly growth factor-dependent and thereforemay not be an integral part of the internal cell cycle controlmechanism, but may occur only in response to external signalling (Scherr1993). The PRAD1 gene, found overexpressed in some parathyroid adenomasis identical to the D1 gene (Motokura et al. 1991). D1 has also beenfound to be over-expressed in a glioblastoma cell line (Xiong et al.1991) and is subject to deregulation by gene amplification (Lammie etal. 1991; Keyomarsi and Pardee 1993). Deregulation of D1 occurs byunknown mechanisms in some lymphomas, squamous cell tumors and breastcarcinomas (Bianchi et al. 1993). This protein is involved in activatingthe growth of cells and therefore, deregulated expression of this geneappears to be an oncogenic event. Some evidence exists that the E-typecyclin may function in the G1 to S transition in human cells: thiscyclin binds to and activates p34cdc2 protein in extracts of humanlymphoid cells in G1, the protein is associated with histone H1 kinaseactivity in HeLa cells (Koff et al. 1991) and cyclin E mRNA isspecifically expressed in late G1 in HeLa cells (Lew et al. 1991).

It has been hypothesized that p34 cdc2 acts at discrete transitionpoints in the cell cycle by phosphorylating varying substrates. Thephosphorylating activity is manifest upon association of the kinase withcyclins which are differentially expressed throughout the cycle of thecell. These different cyclins may alter the substrate specificity of thekinase or may alter its catalytic efficiency (Pines and Hunter 1990).Obvious potential substrates for the cdc2 kinase are transcriptionfactors that control cell-cycle-stage-specific gene transcription.

Disruption of any one of the three CLN genes in yeast does notappreciably affect cell growth, however, upon disruption of all of theCLN genes, cells arrest in G1. In addition, in response to matingpheromone, the CLN proteins are inhibited and yeast cell growth isarrested. Two genes whose products inhibit cyclin activity have beenidentified in S. cerevisiae. The products of the FAR1 and FUS3 genesinhibit CLN2 and CLN3 function, respectively. With pheromone signalling,the levels of Far1p and Fus3p increase, the G1 cyclins do notaccumulate, the CDC28p kinase remains inactive, and cell growth isarrested in G1. These observations suggest that inhibitors of the cyclinproteins, inhibitors of a productive association between the cyclins andthe kinase, or inactivators of the kinase can foster cellular growtharrest.

By contrast, cyclins which are uninhabitable appear to function asuncontrolled positive growth regulators. High level expression of theCLN proteins is a lethal condition in yeast cells. Data indicate thatthe loss of controlled expression of cyclin D1 through chromosomalbreakage, chromosomal translocation or gene amplification can promoteoncogenicity in mammalian cells (Xiong et al. 1991; Lammie et al. 1991;Bianchi et al. 1993). In addition, it appears that events that disruptthe control of cyclin expression and control of cyclin function canresult in bypass of the G1 checkpoint and dysregulated cellular growth.The cyclin proteins which operate in G1 to promote cellularproliferation would be ideal targets for therapeutics aimed at controlof cell growth. Candidate surrogate proteins for substitution in theyeast pathway for identification of such therapeutics include humancyclins C, D and E. All three proteins are normally expressed during theG1 phase of the mammalian cell cycle and are thus candidate mediators ofthe commitment of cells to proliferate.

Examples of compounds which are known to act in G1 to prevent entry intoS phase are transforming growth factor β (TGF-β) and rapamycin.Rapamycin, an immunosuppressant, inhibits the activity of cyclin E-boundkinase. This macrolide acts in G1 to prevent the proliferation ofIL-2-stimulated T lymphocytes (Scherr 1993). TGF-β has been shown toprevent progression from G1 to S phase in mink lung epithelial cells(Howe et al. 1991). TGF-β appears to interfere with activation of thekinase, perhaps by reducing the stability of the complex which thekinase forms with cyclin E (Koff 1993).

A strain of yeast cells bearing inactive CLN1, CLN2 and CLN3 genes andan integrated chimeric gene encoding a Gal1 promoter-driven human CLNsequence (see DL1 cells, Lew et al. Cell 66, 1197 (1991) will serve as atester strain. The Gal1 promoter permits high level expression whencells are grown in the presence of galactose but this promoter isrepressed when cells are grown on glucose. Yeast cells so engineered arenonviable on glucose due to an absence of expression of functionalcyclin. These yeast, however, proliferate on galactose-containing mediumdue to expression of the human cyclin sequence. Exposure of this strainto an inhibitor of cyclin function would render the cells incapable ofgrowth, even on galactose medium, i.e., the cells would growth arrest inthe presence or absence of galactose. This phenotype could serve as anindication of the presence of an exogenously applied cyclin inhibitorbut would not be useful as a screen for the identification of candidateinhibitors from members of a random peptide library. Growth arrest of asubset of cells in an otherwise growing population is useless as anindicator system. Therefore, in order to identify random peptideinhibitors of mammalian cyclins, a two stage screen is envisioned.

A two-hybrid system described by Fields and Song (Nature 340, 245 (1989)permits the detection of protein-protein interactions in yeast. GAL4protein is a potent activator of transcription in yeast grown ongalactose. The ability of GAL4 to activate transcription depends on thepresence of an N-terminal sequence capable of binding to a specific DNAsequence (UAS_(G)) and a C-terminal domain containing a transcriptionalactivator. A sequence encoding a protein, "A", may be fused to thatencoding the DNA binding domain of the GAL4 protein. A second hybridprotein may be created by fusing sequence encoding the GAL4transactivation domain to sequence encoding a protein "B". If protein"A" and protein "B" interact, that interaction serves to bring togetherthe two domains of GAL4 necessary to activate transcription of a UAS_(G)-containing gene. In addition to co-expressing plasmids encoding bothhybrid proteins, yeast strains appropriate for the detection ofprotein-protein interactions using this two-hybrid system would containa GAL1-lacZ fusion to permit detection of transcription from a UAS_(G)sequence. These strains should also be deleted for endogenous GAL4 andfor GAL80, a negative regulator of GAL4.

In a variation of the two-hybrid system just described, the GAL4 DNAbinding domain would be fused to a human cyclin sequence. In addition,oligonucleotides encoding random peptides would be ligated to sequenceencoding the GAL4 transactivation domain. Co-transformation ofappropriate yeast strains with plasmids encoding these two hybridproteins and screening for yeast expressing β-galactosidase would permitidentification of yeast expressing a random peptide sequence capable ofinteracting with a human cyclin. Identification of peptides with thatcapability would be the goal of this first stage of screening. ii. Oncerandom peptides capable of interacting with a human cyclin of interesthad been identified, second stage screening could commence. The secondscreen would permit the identification of peptides that not only boundto human cyclin but, through that interaction, inhibited cyclinactivation of the cell cycle-dependent kinase and, thus, cellularproliferation. Thus, candidate peptides would be expressed,individually, in yeast lacking CLN1, CLN2 and CLN3 but expressing ahuman CLN sequence, as described above. Those peptides, expression ofwhich does not permit growth of the tester strain on galactose, would bepresumed cyclin inhibitors.

An advantage to this two-stage approach to the identification ofpotential cyclin inhibitors is the high probability that random peptidesequences selected in stage one interact with human cyclin proteins. Asubsequently determined ability of that sequence to cause growth arrestof the tester yeast on galactose would be a strong indication that thegrowth arrest was due to a direct effect of the peptide on the cyclinand not on another protein, e.g., the cell cycle dependent kinase.Though a strong indication, such a result would not be an absoluteindication and verification of the inhibitory effect on cyclin functioncould be obtained in vitro through biochemical assay.

Screening and Selection

A marker gene is a gene whose expression causes a phenotypic changewhich is screenable or selectable. If the change is selectable, thephenotypic change creates a difference in the growth or survival ratebetween cells which express the marker gene and those which do not. Ifthe change is screenable, the phenotype change creates a difference insome detectable characteristic of the cells, by which the cells whichexpress the marker may be distinguished from those which do not.Selection is preferable to screening.

The marker gene may be coupled to the yeast pheromone pathway so thatexpression of the marker gene is dependent on activation of the Gprotein. This coupling may be achieved by operably linking the markergene to a pheromone-responsive promoter. The term "pheromone-responsivepromoter" indicates a promoter which is regulated by some product of theyeast pheromone signal transduction pathway, not necessarily pheromoneper se. In one embodiment, the promoter is activated by the pheromonepathway, in which case, for selection, the expression of the marker geneshould result in a benefit to the cell. A preferred marker gene is theimidazoleglycerol phosphate dehydratase gene (HIS3). If a pheromoneresponsive promoter is operably linked to a beneficial gene, the cellswill be useful in screening or selecting for agonists. If it is linkedto a deleterious gene, the cells will be useful in screening orselecting for antagonists.

Alternatively, the promoter may be one which is repressed by thepheromone pathway, thereby preventing expression of a product which isdeleterious to the cell. With a pheromone-repressed promoter, onescreens for agonists by linking the promoter to a deleterious gene, andfor antagonists, by linking it to a beneficial gene.

Repression may be achieved by operably linking a pheromone-inducedpromoter to a gene encoding mRNA which is antisense to at least aportion of the mRNA encoded by the marker gene (whether in the coding orflanking regions), so as to inhibit translation of that mRNA. Repressionmay also be obtained by linking a pheromone-induced promoter to a geneencoding a DNA-binding repressor protein, and incorporating a suitableoperator site into the promoter or other suitable region of the markergene.

Suitable positively selectable (beneficial) genes include the following:URA3, LYS2, HIS3, LEU2, TRP1; ADE1,2,3,4,5,7,8; ARG1,3,4,5,6,8;HIS1,4,5; ILV1,2,5; THR1,4; TRP2,3,4,5; LEU1,4; MET2,3,4,8,9,14,16,19;URA1,2,4,5,10; HOM3,6; ASP3; CHO1; ARO 2,7; CYS3; OLE1; INO1,2,4; PRO1,3Countless other genes are potential selective markers. The above areinvolved in well-characterized biosynthetic pathways.

The imidazoleglycerol phosphate dehydratase (IGP dehydratase) gene(HIS3) is preferred because it is both quite sensitive and can beselected over a broad range of expression levels. In the simplest case,the cell is auxotrophic for histidine (requires histidine for growth) inthe absence of activation. Activation leads to synthesis of the enzymeand the cell becomes prototrophic for histidine (does not requirehistidine). Thus the selection is for growth in the absence ofhistidine. Since only a few molecules per cell of IGP dehydratase arerequired for histidine prototrophy, the assay is very sensitive.

In a more complex version of the assay, cells can be selected forresistance to aminotriazole (AT), a drug that inhibits the activity ofIGP dehydratase. Cells with low, fixed level of expression of HIS3 aresensitive to the drug, while cells with higher levels are resistant. Theamount of AT can be selected to inhibit cells with a basal level of HIS3expression (whatever that level is) but allow growth of cells with aninduced level of expression. In this case selection is for growth in theabsence of histidine and in the presence of a suitable level of AT.

In appropriate assays, so-called counterselectable or negativelyselectable genes may be used. Suitable genes include: URA3(orotidine-5'-phosphate decarboxylase; inhibits growth on 5-fluorooroticacid), LYS2 (2-aminoadipate reductase; inhibits growth on α-aminoadipateas sole nitrogen source), CYH2 (encodes ribosomal protein L29;cycloheximide-sensitive allele is dominant to resistant allele), CAN1(encodes arginine permease; null allele confers resistance to thearginine analog canavanine), and other recessive drug-resistant markers.

The natural response to induction of the yeast pheromone responsepathway is for cells to undergo growth arrest. This is the preferred wayto select for antagonists to a ligand/receptor pair that induces thepathway. An autocrine peptide antagonist would inhibit the activation ofthe pathway; hence, the cell would be able to grow. Thus, the FAR1 genemay be considered an endogenous counterselectable marker. The FAR1 geneis preferably inactivated when screening for agonist activity.

The marker gene may also be a screenable gene. The screenedcharacteristic may be a change in cell morphology, metabolism or otherscreenable features. Suitable markers include beta-galactosidase (Xgal,C₁₂ FDG, Salmon-gal, Magenta-Gal (latter two from Biosynth Ag)),alkaline phosphatase, horseradish peroxidase, exo-glucanase (product ofyeast exb1 gene; nonessential, secreted); luciferase; andchloramphenicol transferase. Some of the above can be engineered so thatthey are secreted (although not β-galactosidase). The preferredscreenable marker gene is beta-galactosidase; yeast cells expressing theenzyme convert the colorless substrate Xgal into a blue pigment. Again,the promoter may be pheromone-induced or pheromone-inhibited.

Yeast Cells

The yeast may be of any species that possess a G protein-mediated signaltransduction pathway and which are cultivatable. Suitable speciesinclude Kluyverei lactis, Schizosaccharomyces pombe, and Ustilagomaydis; Saccharomyces cerevisiae is preferred. Either Gα or Gβγ may bethe activator of the "effecter." (It is suspected that in some species,both Gα-activated and Gβγ-activated effectors exist.) The term "yeast",as used herein, includes not only yeast in a strictly taxonomic sense(i.e., unicellular organisms), but also yeast-like multicellular fungiwith pheromone responses mediated by the mating pathway.

The yeast cells of the present invention may be used to test peptidesfor the ability to interact with an exogenous G protein-coupled receptoror other PSP surrogate. The yeast cells must express both the exogenousG protein-coupled receptor (or other SP surrogate), and a complementaryG protein (or other PSPs necessary for the PSP surrogate to function inthe pheromone system, if need be after activation by a drug), and thesemolecules must be presented in such a manner that a "readout" can beobtained by means of the pheromone response pathway (which may bemodified to improve the readout).

For a readout to be possible, a gene encoding a selectable or screenabletrait must be coupled to the G protein-mediated signal transductionpathway so that the level of expression of the gene is sensitive to thepresence or absence of a signal, i.e., binding to the coupled exogenousreceptor. This gene may be an unmodified gene already in the pathway,such as the genes responsible for growth arrest. It may be a yeast gene,not normally a part of the pathway, that has been operably linked to a"pheromone-responsive" promoter. Or it may be a heterologous gene thathas been so linked. Suitable genes and promoters were discussed above.

It will be understood that to achieve selection or screening, the yeastmust have an appropriate phenotype. For example, introducing apheromone-responsive chimeric HIS3 gene into a yeast that has awild-type HIS3 gene would frustrate genetic selection. Thus, to achievenutritional selection, an auxotrophic strain is wanted.

The yeast cells of the present invention optionally possess one or moreof the following characteristics:

(a) the endogenous FAR1 gene has been inactivated;

(b) the endogenous SST2 gene, and/or other genes involved indesensitization, has been inactivated;

(c) the endogenous pheromone (a- or α-factor) receptor gene has beeninactivated; and

(d) the endogenous pheromone genes have been inactivated.

"Inactivation" means that production of a functional gene product isprevented or inhibited. Inactivation may be achieved by deletion of thegene, mutation of the promoter so that expression does not occur, ormutation of the coding sequence so that the gene product is inactive.Inactivation may be partial or total.

Mutants with inactivated supersensitivity-related genes can beidentified by conventional genetic screening procedures. The far1 genewas identified as an α-factor resistant mutant that remained blue (withfus1-lacZ) on α-factor/Xgal. far2, as it turns out, is the same as fus3.Supersensitive mutants could be identified as constitutive weak bluecolonies expressing fus1-lacZ on Xgal, or as strains that can mate moreproficiently with a poor pheromone-secreter.

The DNA sequences of (a) the α- and a-factor genes, (b) the α- anda-factor receptors, (c) the FAR1 gene, (d) the SST2 gene, and (e) theFUS1 promoter have been reported in the following references:

MFa1 and MFa2: A. J. Brake, C. Brenner, R. Najarian, P. Laybourn, and J.Merryweather. Structure of Genes Encoding Precursors of the YeastPeptide Mating Pheromone a-Factor. In Protein Transport and Secretion.Gething M.-J., ed. Cold Spring Harbor Lab, New York, 1985.

MFα1 and MFα2: Singh, A. E. Y. Chen, J. M. Lugovoy, C. N. Chang, R. A.Hitzeman et al. 1983. Saccharomyces cerevisiae contains two discretegenes coding for the α-pheromone. Nucleic Acids Res. 11:4049; J. Kurjanand I. Herskowitz. 1982. Structure of a yeast pheromone gene (MF): Aputative α-factor precursor contains four tandem copies of matureα-factor. Cell 30:933.

STE2 and STE3: A. C. Burkholder and L. H. Hartwell. 1985. The yeastα-factor receptor: Structural properties deduced from the sequence ofthe STE2 gene. Nucleic Acids Res. 13:8463; N. Nakayama, A. Miyajima, andK. Arai. 1985. Nucleotide sequences of STE2 and STE3, cell type-specificsterile genes from Saccharomyces cerevisiae. EMBO J. 4:2643; D. C.Hagen, G. McCaffrey, and G. F. Sprague, Jr. 1986. Evidence the yeastSTE3 gene encodes a receptor for the peptide pheromone a-factor: Genesequence and implications for the structure of the presumed receptor.Proc Natl Acad Sci 83:1418.

FAR1: F. Chang and I. Herskowitz. 1990. Identification of a genenecessary for cell cycle arrest by a negative growth factor of yeast:FAR1 is an inhibitor of a G1 cyclin, CLN2. Cell 63:999.

SST2: C. Dietzel and J. Kurjan. 1987. Pheromonal regulation and sequenceof the Saccharomyces cerevisiae SST2 gene: A model for desensitizationto pheromone. Mol Cell Biol 7: 4169.

FUS1: J. Trueheart, J. D. Boeke, and G. R. Fink. 1987. Two genesrequired for cell fusion during yeast conjugation: Evidence for apheromone-induced surface protein. Mol Cell Biol 7:2316.

The various essential and optional features may be imparted to yeastcells by, e.g., one or more of the following means: isolation ofspontaneous mutants with one or more of the desired features; mutationof yeast by chemical or radiation treatment, followed by selection; andgenetic engineering of yeast cells to introduce, modify or delete genes.

Other explicit characteristics desirable in strains of yeast designed tobe used as screening devices for inhibitors/activators of PSP surrogatesare discussed in subsections dealing specifically with each moleculartarget.

Peptide

The term "peptide" is used herein to refer to a chain of two or moreamino acids, with adjacent amino acids joined by peptide (--NHCO--)bonds. Thus, the peptides of the present invention includeoligopeptides, polypeptides, and proteins. Preferably, the peptides ofthe present invention are 2 to 200, more preferably 5 to 50, amino acidsin length. The minimum peptide length is chiefly dictated by the need toobtain sufficient potency as an activator or inhibitor. The maximumpeptide length is only a function of synthetic convenience once anactive peptide is identified.

For initial studies in which the cognate PSP was a yeast pheromonereceptor, a 13-amino acid peptide was especially preferred as that isthe length of the mature yeast α-factor.

Peptide Libraries A "peptide library" is a collection of peptides ofmany different sequences (typically more than 1000 different sequences),which are prepared essentially simultaneously, in such a way that, iftested simultaneously for some activity, it is possible to characterizethe "positive" peptides.

The peptide library of the present invention takes the form of a yeastcell culture, in which essentially each cell expresses one, and usuallyonly one, peptide of the library. While the diversity of the library ismaximized if each cell produces a peptide of a different sequence, it isusually prudent to construct the library so there is some redundancy.

In the present invention, the peptides of the library are encoded by amixture of DNA molecules of different sequence. Each peptide-encodingDNA molecule is ligated with a vector DNA molecule and the resultingrecombinant DNA molecule is introduced into a yeast cell. Since it is amatter of chance which peptide-encoding DNA molecule is introduced intoa particular cell, it is not predictable which peptide that cell willproduce. However, based on a knowledge of the manner in which themixture was prepared, one may make certain statistical predictions aboutthe mixture of peptides in the peptide library.

It is convenient to speak of the peptides of the library as beingcomposed of constant and variable residues. If the nth residue is thesame for all peptides of the library, it is said to be constant. If thenth residue varies, depending on the peptide in question, the residue isa variable one. The peptides of the library will have at least one, andusually more than one, variable residue. A variable residue may varyamong any of two to all twenty of the genetically encoded amino acids;the variable residues of the peptide may vary in the same or differentmanner. Moreover, the frequency of occurrence of the allowed amino acidsat a particular residue position may be the same or different. Thepeptide may also have one or more constant residues.

There are two principal ways in which to prepare the required DNAmixture. In one method, the DNAs are synthesized a base at a time. Whenvariation is desired, at a base position dictated by the Genetic Code, asuitable mixture of nucleotides is reacted with the nascent DNA, ratherthan the pure nucleotide reagent of conventional polynucleotidesynthesis.

The second method provides more exact control over the amino acidvariation. First, trinucleotide reagents are prepared, eachtrinucleotide being a codon of one (and only one) of the amino acids tobe featured in the peptide library. When a particular variable residueis to be synthesized, a mixture is made of the appropriatetrinucleotides and reacted with the nascent DNA.

Once the necessary "degenerate" DNA is complete, it must be joined withthe DNA sequences necessary to assure the expression of the peptide, asdiscussed in more detail below, and the complete DNA construct must beintroduced into the yeast cell.

Expression System

The expression of a peptide-encoding gene in a yeast cell requires apromoter which is functional in yeast. Suitable promoters include thepromoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman etal., J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hesset al., J. Adv. Enzyme Reg. 7, 149 (1968); and Holland et al.Biochemistry 17, 4900 (1978)), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. Suitable vectors andpromoters for use in yeast expression are further described in R.Hitzeman et al., EPO Publn. No. 73,657. Other promoters, which have theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, and the aforementioned metallothionein andglyceraldehyde-3-phosphate dehydrogenase, as well as enzymes responsiblefor maltose and galactose utilization. Finally, promoters that areactive in only one of the two haploid mating types may be appropriate incertain circumstances. Among these haploid-specific promoters, thepheromone promoters MFa1 and MFα1 are of particular interest.

In screens devised for a subset of PSP surrogates (e.g. kinases,cyclins) random peptide sequences need not be expressed in the contextof yeast pheromone and need not be engineered for secretion or transportto the extracellular space. Libraries of random peptides may beexpressed in a multiplicity of ways, including as portions of chimericproteins, as in a two-hybrid protein system designed to signalprotein-protein interactions. Random peptides need not necessarilysubstitute for yeast pheromones but can impinge on the pheromone pathwaydownstream of the interaction between pheromone and pheromone receptor(as in random peptide inhibitors of the kinases or of the cyclins).

In constructing suitable expression plasmids, the termination sequencesassociated with these genes, or with other genes which are efficientlyexpressed in yeast, may also be ligated into the expression vector 3' ofthe heterologous coding sequences to provide polyadenylation andtermination of the mRNA.

Vectors

The vector must be capable of replication in a yeast cell. It may be aDNA which is integrated into the host genome, and thereafter isreplicated as a part of the chromosomal DNA, or it may be DNA whichreplicates autonomously, as in the case of a plasmid. In the lattercase, the vector must include an origin of replication which isfunctional in the host. In the case of an integrating vector, the vectormay include sequences which facilitate integration, e.g., sequenceshomologous to host sequences, or encoding integrases.

Besides being capable of replication in yeast cells, it is convenient ifthe vector can also be replicated in bacterial cells, as many geneticmanipulations are more conveniently carried out therein. Shuttle vectorscapable of replication in both yeast and bacterial cells include YEps,YIps, and the pRS series.

Periplasmic Secretion

The cytoplasm of the yeast cell is bounded by a lipid bilayer called theplasma membrane. Between this plasma membrane and the cell wall is theperiplasmic space. Peptides secreted by yeast cells cross the plasmamembrane through a variety of mechanisms and thereby enter theperiplasmic space. The secreted peptides are then free to interact withother molecules that are present in the periplasm or displayed on theouter surface of the plasma membrane. The peptides then either undergore-uptake into the cell, diffuse through the cell wall into the medium,or become degraded within the periplasmic space.

The peptide library may be secreted into the periplasm by one of twodistinct mechanisms, depending on the nature of the expression system towhich they are linked. In one system, the peptide may be structurallylinked to a yeast signal sequence, such as that present in the α-factorprecursor, which directs secretion through the endoplasmic reticulum andGolgi apparatus. Since this is the same route that the receptor proteinfollows in its journey to the plasma membrane, opportunity exists incells expressing both the receptor and the peptide library for aspecific peptide to interact with the receptor during transit throughthe secretory pathway. This has been postulated to occur in mammaliancells exhibiting autocrine activation. Such interaction would likelyyield activation of the linked pheromone response pathway duringtransit, which would still allow identification of those cellsexpressing a peptide agonist. For situations in which peptideantagonists to externally applied receptor agonist are sought, thissystem would still be effective, since both the peptide antagonist andreceptor would be delivered to the outside of the cell in concert. Thus,those cells producing an antagonist would be selectable, since thepeptide antagonist would be properly and timely situated to prevent thereceptor from being stimulated by the externally applied agonist.

An alternative mechanism for delivering peptides to the periplasmicspace is to use the ATP-dependent transporters of the STE6/MDR1 class.This transport pathway and the signals that direct a protein or peptideto this pathway are not as well characterized as is the endoplasmicreticulum-based secretory pathway. Nonetheless, these transportersapparently can efficiently export certain peptides directly across theplasma membrane, without the peptides having to transit the ER/Golgipathway. We anticipate that at least a subset of peptides can besecreted through this pathway by expressing the library in context ofthe a-factor prosequence and terminal tetrapeptide. The possibleadvantage of this system is that the receptor and peptide do not comeinto contact until both are delivered to the external surface of thecell. Thus, this system strictly mimics the situation of an agonist orantagonist that is normally delivered from outside the cell. Use ofeither of the described pathways is within the scope of the invention.

The present invention does not require periplasmic secretion, or, ifsuch secretion is provided, any particular secretion signal or transportpathway.

EXAMPLE 1

Development of Autocrine Yeast Strains

In this example, we describe a pilot experiment in which haploid cellswere engineered to be responsive to their own pheromones (see FIG. 1).(Note that in the examples, functional genes are capitalized andinactivated genes are in lower case.) For this purpose we constructedrecombinant DNA molecules designed to:

i. place the coding region of STE2 under the transcriptional control ofelements which normally direct the transcription of STE3. This is donein a plasmid that allows the replacement of genomic STE3 of S.cerevisiae with sequences wherein the coding sequence of STE2 is drivenby STE3 transcriptional control elements.

ii. place the coding region of STE3 under the transcriptional control ofelements which normally direct the transcription of STE2. This is donein a plasmid which will allow the replacement of genomic STE2 of S.cerevisiae with sequences wherein the coding sequence of STE3 is drivenby STE2 transcriptional control elements.

The sequence of the STE2 gene is known see Burkholder A. C. and HartwellL. H. (1985), "The yeast α-factor receptor: Structural propertiesdeduced from the sequence of the STE2 gene," Nuc. Acids Res. 13, 8463;Nakayama N., Miyajima A., Arai K. (1985) "Nucleotide sequences of STE2and STE3, cell type-specific sterile genes from Saccharomycescerevisiae," EMBO J. 4, 2643.

A 4.3 kb BamHI fragment that contains the entire STE2 gene was excisedfrom plasmid YEp24-STE2 (obtained from J. Thorner, Univ. of California)and cloned into PALTER (Protocols and Applications Guide, 1991, PromegaCorporation, Madison, Wis.). An SpeI site was introduced 7 nucleotides(nts) upstream of the ATG of STE2 with the following mutagenicoligonucleotide, using the STE2 minus strand as template (mutated basesare underlined and the start codon is in bold type):

                          (SEQ ID NO:14)                                          5' GTTAAGAACCATATACTAGTATCAAAAATGTCTG 3'.                                 

A second SpeI site was simultaneously introduced just downstream of theSTE2 stop codon with the following mutagenic oligonucleotide (mutatedbases are underlined and the stop codon is in bold type):

                          (SEQ ID NO:15)                                          5' TGATCAAAATTTACTAGTTTGAAAAAGTAATTTCG 3'.                                

The BamHI fragment of the resulting plasmid (Cadus 1096), containingSTE2 with SpeI sites immediately flanking the coding region, was thensubcloned into the yeast integrating vector YIp19 to yield Cadus 1143.

The STE3 sequence is also known. Nakayama N., Miyajima A., Arai K.(1985), "Nucleotide sequences of STE2 and STE3, cell type-specificsterile genes from Saccharomyces cerevisiae," EMBO J. 4, 2643; Hagen D.C., McCaffrey G., Sprague G. F. (1986), "Evidence the yeast STE3 geneencodes a receptor for the peptide pheromone a-factor: gene sequence andimplications for the structure of the presumed receptor," Proc. Natl.Acad. Sci. 83, 1418. STE3 was made available by Dr. J. Broach as a 3.1kb fragment cloned into PBLUESCRIPT-KS II (Stratagene, 11011 NorthTorrey Pines Road, La Jolla, Calif. 92037). STE3 was subcloned as aKpnI-XbaI fragment into both M13mp18 RF (to yield Cadus 1105) and pUC19(to yield Cadus 1107). The two SpeI sites in Cadus 1107 were removed bydigestion with SpeI, fill-in with DNA polymerase I Klenow fragment, andrecircularization by blunt-end ligation. Single-stranded DNA containingthe minus strand of STE 3 was obtained using Cadus 1105 and SpeI siteswere introduced 9 nts upstream of the start codon and 3 nts downstreamof the stop codon of STE3 with the following mutagenic oligonucleotides,respectively:

                          (SEQ ID NO:16)                                          5' GGCAAAATACTAGTAAAATTTTCATCTC 3'.                                                                 (SEQ ID NO:17)                                          5' GGCCCTTAACACACTAGTGTCGCATTATATTTAC 3'.                                 

The mutagenesis was accomplished using the T7-GEN protocol of UnitedStates Biochemical (T7-GEN In Vitro Mutagenesis Kit, Descriptions andProtocols, 1991, United States Biochemical, P.O. Box 22400, Cleveland,Ohio 44122). The replicative form of the resulting Cadus 1141 wasdigested with AflII and KpnI, and the approximately 2 kb fragmentcontaining the entire coding region of STE3 flanked by the two newlyintroduced Spe I sites was isolated and ligated with the approximately3.7 kb vector fragment of AflII- and KpnI-digested Cadus 1107, to yieldCadus 1138. Cadus 1138 was then digested with XbaI and KpnI, and theSTE3-containing 2.8 kb fragment was ligated into the XbaI- andKpnI-digested yeast integrating plasmid pRS406 (Sikorski, R. S. andHieter, P. (1989) "A System of Shuttle Vectors and Yeast Host StrainsDesigned for Efficient Manipulation of DNA in Saccharomyces cerevisiae",Genetics 122:19-27 to yield Cadus 1145.

The SpeI fragment of Cadus 1143 was replaced with the SpeI fragment ofCadus 1145 to yield Cadus 1147, in which the coding sequences of STE3are under the control of STE2 expression elements. Similarly, the SpeIfragment of Cadus 1145 was replaced with the SpeI fragment of Cadus 1143to yield Cadus 1148, in which the coding sequences of STE2 are under thecontrol of STE3 expression elements. Using the method of pop-in/pop-outreplacement (Rothstein, R. (1991) "[19] Targeting, Disruption,Replacement, and Allele Rescue: Integrative DNA Transformation inYeast", Methods in Enzymology, 194:281-301), Cadus 1147 was used toreplace genomic STE2 with the ste2-STE3 hybrid in a MATa cell and Cadus1148 was used to replace genomic STE3 with the ste3-STE2 hybrid in aMATα cell. Cadus 1147 and 1148 contain the selectable marker URA3.

Haploid yeast of mating type a which had been engineered to express HIS3under the control of the pheromone-inducible FUS1 promoter weretransformed with CADUS 1147, and transformants expressing URA3 wereselected. These transformants, which express both Ste2p and Ste3p, wereplated on 5-fluoroorotic acid to allow the selection of clones which hadlost the endogenous STE2, leaving in its place the heterologous,integrated STE3. Such cells exhibited the ability to grow on mediadeficient in histidine, indicating autocrine stimulation of thepheromone response pathway.

Similarly, haploids of mating type α that can express HIS3 under thecontrol of the pheromone-inducible FUS1 promoter were transformed withCADUS 1148 and selected for replacement of their endogenous STE3 withthe integrated STE2. Such cells showed, by their ability to grow onhistidine-deficient media, autocrine stimulation of the pheromoneresponse pathway.

EXAMPLE 2

Strain Development

In this example, yeast strains are constructed which will facilitateselection of clones which exhibit autocrine activation of the pheromoneresponse pathway. To construct appropriate yeast strains, we will use:the YIp-STE3 and pRS-STE2 knockout plasmids described above, plasmidsavailable for the knockout of FAR1, SST2, and HIS3, and mutant strainsthat are commonly available in the research community. The followinghaploid strains will be constructed, using one-step or two-step knockoutprotocols described in Meth. Enzymol 194:281-301, 1991:

    ______________________________________                                        1. MATα                                                                        ste3::STE2::ste3                                                                          far1   sst2 FUS1::HIS3                                     2. MATa                                                                              ste2::STE3::ste2                                                                          far1   sst2 FUS1::HIS3                                     3. MATα                                                                        ste3::STE2::ste3                                                                          far1   sst2 mfα1                                                                         mfα2                                                                         FUS1::HIS3                           4. MATa                                                                              ste2::STE3::ste2                                                                          far1   sst2 mfa1 mfa2 FUS1::HIS3                           5. MATa bar1 far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3                 6. MATa mfa1 mfa2 far1-1 his3::fus1-HIS3 ste2-STE3 ura3 met1                  ade1 leu2                                                                     ______________________________________                                    

Strains 1 and 2 will be tested for their ability to grow onhistidine-deficient media as a result of autocrine stimulation of theirpheromone response pathways by the pheromones which they secrete. Ifthese tests prove successful, strain 1 will be modified to inactivateendogenous MFα1 and MFα2. The resulting strain 3, MATα far1 sst2ste3::STE2::ste3 FUS1::HIS3 mfa1 mfa2, should no longer display theselectable phenotype (i.e., the strain should be auxotrophic forhistidine). Similarly, strain 2 will be modified to inactivateendogenous MFa1 and MFa2. The resulting strain 4, MATa far1 sst2ste2::STE3::ste2 FUS1::HIS3 mfa1 mfa2, should be auxotrophic forhistidine. The uses of strains 5 and 6 are outlined in Examples 3 and 4below.

EXAMPLE 3

Peptide Library

In this example, a synthetic oligonucleotide encoding a peptide isexpressed so that the peptide is secreted or transported into theperiplasm.

i. The region of MFα1 which encodes mature α-factor has been replacedvia single-stranded mutagenesis with restriction sites that can acceptoligonucleotides with AflII and BglII ends. Insertion ofoligonucleotides with AflII and BglII ends will yield plasmids whichencode proteins containing the MFα1 signal and leader sequences upstreamof the sequence encoded by the oligonucleotides. The MFα1 signal andleader sequences should direct the processing of these precursorproteins through the pathway normally used for the transport of matureα-factor.

The MFα1 gene, obtained as a 1.8 kb EcoRI fragment from pDA6300 (J.Thorner, Univ. of California) was cloned into pALTER (see FIG. 2) inpreparation for oligonucleotide-directed mutagenesis to remove thecoding region of mature α-factor while constructing sites for acceptanceof oligonucleotides with AflII and BclI ends. The mutagenesis wasaccomplished using the minus strand as template and the followingmutagenic oligonucleotide:

    5'CTAAAGAAGA AGGGGTATCT TTGCTTAAGC TCGAGATCTC GACTGATAAC AACAGTGTAG 3'        (SEQ ID NO:18).                                                           

A HindIII site was simultaneously introduced 7 nts upstream of the MFα1start codon with the oligonucleotide:

                          (SEQ ID NO:19)                                          5'CATACACAAT ATAAAGCTTT AAAAGAATGA G 3'.                                  

The resulting plasmid, Cadus 1214, contains a HindIII site 7 ntsupstream of the MFα1 initiation codon, an AflII site at the positionswhich encode the KEX2 processing site in the MFα1 leader peptide, andXhoI and BglII sites in place of all sequences from the leader-encodingsequences up to and including the normal stop codon. The 1.5 kb HindIIIfragment of Cadus 1214 therefore provides a cloning site foroligonucleotides to be expressed in yeast and secreted through thepathway normally travelled by endogenous α-factor.

A sequence comprising the ADH1 promoter and 5' flanking sequence wasobtained as a 1.5 kb BamHI-HindIII fragment from pAAH5 (Ammerer, G.(1983) "[11] Expression of Genes in Yeast Using the ADCI Promoter",Academic Press, Inc., Meth. Enzymol. 101, 192-201 and ligated into thehigh copy yeast plasmid pRS426 (Christianson, T. W. et al. (1992)"Multifunctional yeast high-copy-number shuttle vectors", Gene110:119-122) (see FIG. 3). The unique XhoI site in the resulting plasmidwas eliminated to yield Cadus 1186. The 1.5 Kb HindIII fragment of Cadus1214 was inserted into HindIII-digested Cadus 1186; expression ofsequences cloned into this cassette initiates from the ADH1 promoter.The resulting plasmid, designated Cadus 1215, can be prepared to acceptoligonucleotides with AflII and BclI ends by digestion with thoserestriction endonucleases. The oligonucleotides will be expressed in thecontext of MFα1 signal and leader peptides (FIG. 4).

Modified versions of Cadus 1215 were also constructed. To improve theefficiency of ligation of oligonucleotides into the expression vector,Cadus 1215 was restricted with KpnI and religated to yield Cadus 1337.This resulted in removal of one of two HindIII sites. Cadus 1337 waslinearized with HindIII, filled-in, and recircularized to generate Cadus1338. To further tailor the vector for library construction, thefollowing double-stranded oligonucleotide was cloned into AflII- andBglII-digested Cadus 1338:

                    oligo 062(SEQ ID NO:37)                                       5' TTAAGCGTGAGGCAGAAGCTTATCGATA                                                               oligo 062(SEQ ID NO:38)                                       3'     CGCACTCCGTCTTCGAATAGCTATCTAG                                       

The HindIII site is italicized and a ClaI site is emboldened; this ClaIsite is unique in the resulting vector, Cadus 1373. In Cadus 1373, theHindIII site that exists at the junciton between the MFα pro sequenceand the mature peptide to be expressed by this vector was made unique.Therefore the HindIII site and the downstream BglII site can be used toinsert oligo-nucleotides encoding peptides of interest. Thesemodifications of Cadus 1215 provide an laternative to the use of theAflII site in the cloning of oligonucleotides into the expressionsvector.

Cadus 1373 was altered further to permit elimination from restrictedvector preparations of contaminating singly-cut plasmid. Suchcontamination could result in unacceptably high backgroundtransformation. To eliminate this possibility, approximately 1.1 kb ofdispensable ADH1 sequence at the 51 side of the promoter region wasdeleted. This was accomplished by restruction of Cadus 1373 with SphIand BamHI, fill-in, and ligation; this maneuver regenerates the BamHIsite. The resulting vector, Cadus 1624, was then restricted with HindIIIand ClaI and an approximately 1.4 kb HindIII and ClaI fragment encodinglacZ was inserted to generate Cadus 1625. Use of HindIII- andBglII-restricted Cadus 1625 for acceptance of oligonucleotides resultsin a low background upon transformation of the ligation product intobacteria.

Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BclI, can be ligated into the polylinker of theexpression vector, Cadus 1215. The two single-stranded oligonucleotideshave the following sequences:

    5' G CTA CTT AAG CGT GAG GCA GAA GCT 3'                    (SEQ ID NO:20)

and

    5' C GGA TGA TCA (NNN).sub.n AGC TTC TGC CTC ACG CTT AAG TAG C 3' (SEQ ID NO:21)

where N is any chosen nucleotide and n is any chosen integer. Yeasttransformed with the resulting plasmids will secrete--through theα-factor secretory pathway--peptides whose amino acid sequence isdetermined by the particular choice of N and n (FIG. 4).

Alternatively, the following single stranded oligonucleotides are used:

    MFαNNK (76 mer):                                                        5'CTGGATGCGAAGATCAGCTNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKNNKTGATCAGTCTGTGACGC     3'                                            (SEQ ID NO:39)                 and                                                                           MFαMbo (17 mer):                                                        5' GCGTCACAGACTGATCA 3'                        (SEQ ID NO:40)             

When annealed the double stranded region is:

    TGATCAGTCTGTGACGC (last 17 bases of SEQ ID NO:39)                             ACTAGTCAGACACTGCG (SEQ ID NO:40 in 3' to                                      5' direction)                                                             

where the FokI site is underlined, the BbsI site is emboldened, and theMboI site is italicized. After fill-in using Taq DNA polymerase (PromegaCorporation, Madison, Wis.), the double stranded product is restrictedwith BbsI and MboI and ligated to HindIII- and BglII-restricted Cadus1373.

ii. The region of MFa1 which encodes mature a-factor will be replacedvia single stranded mutagenesis with restriction sites that can acceptoligonucleotides with XhoI and AflII ends. Insertion of oligonucleotideswith XhoI and AflII ends will yield plasmids which encode proteinscontaining the MFa1 leader sequences upstream of the sequence encoded bythe oligonucleotides. The MFa1 leader sequences should direct theprocessing of these precursor proteins through the pathway normally usedfor the transport of mature a-factor.

MFA1, obtained as a BamHI fragment from pKK1 (provided by J. Thorner andK. Kuchler), was ligated into the BamHI site of pALTER (Promega) (FIG.5). Using the minus strand of MFA1 as template, a HindIII site wasinserted by oligonucleotide-directed mutagenesis just 5' to the MFA1start codon using the following oligonucleotide:

                          (SEQ ID NO:22)                                          5' CCAAAATAAGTACAAAGCTTTCGAATAGAAATGCAACCATC.                             

A second oligonucleotide was used simultaneously to introduce a shortpolylinker for later cloning of synthetic oligonucleotides in place ofMFA1 sequences. These MFA1 sequences encode the C-terminal 5 amino acidsof the 21 amino acid leader peptide through to the stop codon:

    5'GCCGCTCCAAAAGAAAAGACCTCGAGCTCGCTTAAGTTCTGCGTACA AAAACGTTGTTC 3' (SEQ ID     NO:23).                                                                   

The 1.6 kb HindIII fragment of the resulting plasmid, Cadus 1172,contains sequences encoding the MFA1 start codon and the N-terminal 16amino acids of the leader peptide, followed by a short polylinkercontaining XhoI, SacI, and AflII sites for insertion ofoligonucleotides. The 1.6 kb HindIII fragment of Cadus 1172 was ligatedinto HindIII-digested Cadus 1186 (see above) to place expression ofsequences cloned into this cassette under the control of the ADH1promoter. The SacI site in the polylinker was made unique by eliminatinga second SacI site present in the vector. The resulting plasmid,designated Cadus 1239, can be prepared to accept oligonucleotides withXhoI and AflII ends by digestion with those restriction endonucleasesfor expression in the context of MFa1 leader peptides (FIG. 6).

Two single-stranded oligonucleotide sequences (see below) aresynthesized, annealed, and repetitively filled in, denatured, andreannealed to form double-stranded oligonucleotides that, when digestedwith AflII and BglII, can be cloned into the polylinker of theexpression vector, Cadus 1239. The two single-stranded oligonucleotidesused for the cloning have the following sequences:

    5' GG TAC TCG AGT GAA AAG GAC AAC 3'          (SEQ ID NO:24)                  5' CG TAC TTA AGC AAT AAC ACA (NNN).sub.n  GTT GTC CTT CTT TTC ACT CGA        GTA CC 3'                                     (SEQ ID NO:25)              

where N is any chosen nucleotide and n is any chosen integer

Yeast transformed with the resulting plasmids will transport

through the pathway normally used for the export of a-factor

farnesylated, carboxymethylated peptides whose amino acid sequence isdetermined by the particular choice of N and n (FIG. 6).

EXAMPLE 4

Peptide Secretion/Transport

This example demonstrates the ability to engineer yeast such that theysecrete or transport oligonucleotide-encoded peptides (in this casetheir pheromones) through the pathways normally used for the secretionor transport of endogenous pheromones.

Autocrine MATa strain CY588

A MATa strain designed for the expression of peptides in the context ofMFα1 (i.e., using the MFα1 expression vector, Cadus 1215) has beenconstructed. The genotype of this strain, which we designate CY588, isMATa bar1 far1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3. The bar1mutation eliminates the strain's ability to produce a protease thatdegrades α-factor and that may degrade some peptides encoded by thecloned oligonucleotides; the far1 mutation abrogates the arrest ofgrowth which normally follows stimulation of the pheromone responsepathway; an integrated FUS1-HIS3 hybrid gene provides a selectablesignal of activation of the pheromone response pathway; and, finally,the ste14 mutation lowers background of the FUS1-HIS3 readout. Theenzymes responsible for processing of the MFα1 precursor in MATα cellsare also expressed in MATa cells (Sprague and Thorner, in The Molecularand Cellular Biology of the Yeast Saccharomyces: Gene Expression, 1992,Cold Spring Harbor Press), therefore, CY588 cells should be able tosecrete peptides encoded by oligonucleotides expressed from plasmidCadus 1215.

A high transforming version (tbtl-1) of CY588 was obtained by crossingCY1013 (CY588 containing an episomal copy of the STE14 gene) (MATabarl::hisGfarl-1 fusl-HIS3 ste14::TRPl ura3 trpl leu2 his3 [STE14 URA3CEN4]) to CY793 (MATα tbtl-1 ura3 leu2 trpl his3 fus1-HIS2 can1stell4::TRP1 [FUS1 LEU2 2μ]) and selecting from the resultant spores astrain possessing the same salient genotype described for CY588 (seeabove), and in addition the tb1-1 allele, which confers the capacity forvery high efficiency transformation by electroporation. The selectedstrain is CY1455 (MATabar1::hisGfar1-1 fus1-HIS3 ste14::TRP1 tbt-1 ura3trp1 leu2 his3).

Secretion of peptides in the context of yeast α-factor

Experiments were performed to test: 1. the ability of Cadus 1215 tofunction as a vector for the expression of peptides encoded by syntheticoligonucleotides; 2. the suitability of the oligonucleotides, asdesigned, to direct the secretion of peptides through the α-factorsecretory pathway; 3. the capacity of CY588 to secrete those peptides;and 4. the ability of CY588 to respond to those peptides that stimulatethe pheromone response pathway by growing on selective media. Theseexperiments were performed using an oligonucleotide which encodes the 13amino acid α-factor; i.e., the degenerate sequence (NNN)_(n) in theoligonucleotide cloned into Cadus 1215 (see above) was specified (n=13)to encode this pheromone. CY588 was transformed with the resultingplasmid (Cadus 1219), and transformants selected on uracil-deficientmedium were transferred to histidine-deficient medium supplemented witha range of concentrations of aminotriazole (an inhibitor of the HIS3gene product that serves to reduce background growth). The results,shown in FIG. 7, demonstrate that the synthetic oligonucleotide,expressed in the context of MFα1 by Cadus 1215, conferred upon CY588 anability to grow on histidine-deficient media supplemented withaminotriazole. In summation, these data indicate that: 1. CY588 iscompetent for the secretion of a peptide encoded by the (NNN)_(n)sequence of the synthetic oligonucleotide cloned into and expressed fromCadus 1215; and 2. CY588 can, in an autocrine fashion, respond to asecreted peptide which stimulates its pheromone response pathway, inthis case by α-factor binding to STE2.

Additional experiments were performed to test the utility of autocrineyeast strains in identifying agonists of the Ste2 receptor from amongmembers of two semi-random a-factor libraries, α-Mid-5 and MFα-8

α-Mid-5 Library

A library of semi-random peptides, termed the α-Mid-5 library, wasconstructed. In this library, the N-terminal four amino acids and theC-terminal four amino acids of a 13 residue peptide are identical tothose of native α-factor while the central five residues (residues 5-9)are encoded by the degenerate sequence (NNQ)₅. The followingoligonucleotides were used in the construction of the α-Mid-5 library:

    (1)MFαMbo, a 17 mer:                                                    5' GCGTCACAGACTGATCA                         (SEQ ID NO:40)                   (2)MID5ALF, a 71 mer:                                                         5' GCCGTCAGTAAAGCTTGGCATTGGTTGNNQNNQNNQNNQMMQCAGCCTATGTACTGATCAGTCTGTGACGC                                                 (SEQ ID NO:41)               

Sequenase (United States Biochemical Corporation, Cleveland, Ohio) wasused to complete the duplex formed after annealing MFαMbo to the MID5ALFoligonucleotide. In the MID5ALF sequence, N indicates a mixture of A, C,G, and T at ratios of 0.8:1:1.3:1; Q indicates a mixture of C and G at aratio of 1:1.3. These ratios were employed to compensate for thedifferent coupling efficiences of the bases during oligonucleotidesynthesis and were thus intended to normalize the appearance of allbases in the library. In the oligonucleotide sequences above, theHindIII site is underlined and the MboI site is emboldened. Thedouble-stranded oligonucleotide was restricted with HindIII and MboI andligated to Cadus 1625 (see above); Cadus 1625 had been prepared toaccept the semi-random oligonucleotides by restriction with HindIII andBglII.

The apparant complexity of the αMid-5 library is 1×10⁷. This complexityis based on the number of bacterial transformants obtained with thelibrary DNA versus transformants obtained with control vector DNA thatlacks insert. Sequence analysis of six clones from the librarydemonstrated that each contained a unique insert.

To identify peptide members of the α-Mid-5 library that could act asagonists on the STE2 receptor, CY1455, a high transforming version ofCY588, was electroporated to enhance uptake of α-Mid-5 DNA.Transformants were selected on uracil-deficient (-Ura) syntheticcomplete medium and were transferred to histidine-deficient (-His)synthetic complete medium supplemented with 0.5 mM or 1 mMaminotriazole.

Yeast able to grow on -His+aminotriazole medium include (1) yeast whichare dependent on the expression of an α-factor variant agonist and (2)yeast which contain mutations that result in constitutive signallingalong the pheromone pathway. Yeast expressing and secreting a variantSTE2 receptor agonist have the ability to stimulate the growth on -Hismedium of surrounding CY 1455 cells that do not express such an agonist.Thus a recognizable formation (termed a "starburst") results, consistingof a central colony, growing by virtue of autocrine stimuation of thepheromone pathway, surrounded by satellite colonies, growing by virtueof paracrine stimulation of the pheromone pathway by the agonist peptideas that ptpetide diffuses radially from the central, secreting colony.

In order to identify the peptide sequence responsible for this"starburst" phenomenon, yeast were transferred from the center of the"starburst" and streaks were made on -Ura medium to obtain singlecolonies. Individual clones from -Ura were tested for the His⁺ phenotypeon -His+aminotriazole plates containing a sparse lawn of CY1455 cells.Autocrine yeast expressing a peptide agonist exhibited the "starburst"phenotype as the secreted agonist stimulated the growth of surroundingcells that lacked the peptide but were capable of responding to it.Constitutive pheromone pathway mutants were capable of growth on-His+aminotriazole but were incapable of enabling the growth ofsurrounding lawn cells.

Alternatively, streaks of candidate autocrine yeast clones were made onplates containing 5-fluoroorotic acid (FOA) to obtain Ura⁻ segregantswere retested on -His+aminotriazole for the loss of the His⁺ phenotype.Clones that lost the ability to grow on -His+aminotriazole afterselection on FOA (and loss of the peptide-encoding plasmid) derived fromcandidate expressors of a peptide agonist. The plasmid was rescued fromcandidate clones and the peptide sequences determined. In addition, aplasmid encoding a putative Ste2 agonist was reintroduced into CY1455 toconfirm that the presence of the plasmid encoding the peptide agonistconferred the His⁺ phenotype to CY1455.

By following the above protocol novel Ste2 agonists have been identifiedfrom the α-Mid-5 library. Sequences of nine agonists follow, preceded bythe sequence fo the native α-factor pheromone and by the oligonucleotideused to encode the native pheromone in these experiments. (Note thevariant codons used in the α-factor-encoding oligonucleotide forglutamine and proline in the C-terminal amino acids of α-factor). Beloweach nucleotide sequence is the encoded amino acid sequence withvariations from the native-pheromone underlined.

    α-factor                                                                     TGG CAT TGG TTG CAG CTA AAA CAA CCA ATG TAC                              encodes                                                                            Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr                           (DNA, SEQ ID NO:42, AAS, SEQ ID NO:43)                                   α-factor                                                                oligo:                                                                             TGG CAT TGG TTG CAG CTA AAACAG CCT ATG TAC                               encodes                                                                            Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pro Met Tyr                      M1   TGG CAT TGG TTG TCC TTG TCG CCC GGG CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Ser Leu Ser Pro Gly Gln Pro Met Tyr                      M2   TGG CAT TGG TTG TCC CTG GAC GCT GGC CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Ser Leu Asp Ala Gly Gln Pro Met Tyr                      M3   TGG CAT TGG TTG ACC TTG ATG GCC GGG CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Thr Leu Met Ala Gly Gln Pro Met Tyr                      M4   TGG CAT TGG TTG CAG CTG TCG GCG GGC CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Gln Leu Ser Ala Gly Gln Pro Met Tyr                      M5   TGG CAT TGG TTG AGG TTG CAG TCC GGC CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Arg Leu Gln Ser Gly Gln Pro Met Tyr                      M6   TGG CAT TGG TTG CGC TTG TCC GCC GGG CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Arg Leu Ser Ala Gly Gln Pro Met Tyr                      M7   TGG CAT TGG TTG TCG CTC GTC CCG GGG CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Ser Leu Val Pro Gly Gln Pro Met Tyr                      M8   TGG CAT TGG TTG TCC CTG TAC CCC GGG CAG CCT ATG TAC                      encodes                                                                            Trp His Trp Leu Ser Leu Tyr Pro Gly Gln Pro Met Tyr                      M9   TGG CAT TGG TTG CGG CTG CAG CCC GGG CAG CCT ATG TAC                      encodes                                                                             Trp His Trp Leu Arg Leu Gln Pro Gly Gln Pro Met Tyr                 

The nine peptide agonists of the Ste2 receptor above were derived fromone electroporation of CY1455 using 1 μg of the α-Mid-5 library DNA.Approximately 3×10⁵ transformants were obtained, representingapproximately 0.03% of the sequences present in that library.

MFα-8 Library

A semi-random α-factor library was obtained through synthesis ofmutagenized α-factor oligonucleotides such that 1 in 10,000 peptideproducts were expected to be genuine α-factor. The mutagenesis wasaccomplished with doped synthesis of the oligonucleotides: eachnucleotide was made approximately 68% accurate by synthesizing thefollowing two oligos:

                                        (SEQ ID NO:44)                            5' CTGGATGCGA AGACTCAGCT (20 mer)(oligo060)                               

where the FokI site is underlined and the BbsI site is emboldened

                                              (69 mer)(oligo074) (SEQ ID                                                    NO:45)                              5' CGGATGATCA gta cat tgg ttg gcc agg ttt tag ctg caa cca atg cca AGC TGA     TC TTC GCA TCC AG                                                         

where the BclI site is italicized, the FokI site is underlined, the BbsIsite is emboldened. The lower case letters indicate a mixture of 67% ofthat nucleotide and 11% of each of the other three nucleotides (e.g. tindicates 67% and 11% A, 11% C, and 11% G). Note that digestion of thedouble-stranded oligonucleotide by FokI or BbsI will yield an identical5' end that is compatible with HindIII ends.

Oligos 060 and 074 will form the following double-stranded molecule whenannealed:

    5' CTGGATGCGAAGACCTCAGC T                                                     3' GACCTACGTTCTGAGTCGA acc gta acc aac gtc gat ttt gga ccg gtt ggt tac        atg ACTAGTAGGC 5'                                                         

The duplex was repetitively filled-in using Taq DNA polymerase (PromegaCorporation, Madison, Wis.). The double-stranded product was restrictedwith BbsI and BclI and ligated into HindIII- and BglII-digested Cadus1373. The BglII/BclI joint creates a TGA stop codon for the terminationof translation of the randomers. Using this approach, the MFα-5.8library (a library of apparent low complexity based on PCR analysis ofoligonucleotide insert frequency) was constructed. To identify peptidemembers of the MFα-5.8 library that could act as agonists on the STE2receptor, CY1455, a high transforming version of CY588, waselectroporated to enhance uptake of MFα-5.8 DNA. Transformants wereselected on uracil-deficient (-Ura) synthetic complete medium and weretransferred to histidine-deficient (-His) synthetic complete mediumsupplemented with 1.0 mM or 2.5 mM aminotriazole. Yeast from colonieswhich were surrounded by satellite growth were transferred as streaks to-Ura medium to obtain single colonies. Yeast from single colonies wreethen tested for the His⁺ phenotype on -His+aminotriazole plates.Sequence analysis of seven of the plasmids rescued from His⁺ yeastrevealed three unique α-factor variants that acted as agonists on theSTE2 receptor.

1.4 independent clones had the following sequence:

    1.4 independent clones had the following sequence:                                      TGG CAT TGG CTA CAG CTA ACG CCT GGG CAA CCA ATG TAC  (SEQ ID        NO:46)                                                                        encoding  Trp His Trp Leu Gln Leu Thr Pro Gly Gln Pro Met Tyr  (SEQ ID        NO:47)                                                                        2.2 independent clones had the following sequence:                                      TGG CAT TGG CTG GAG CTT ATG CCT GGC CAA CCA TTA TAC  (SEQ ID        NO:48)                                                                        encoding  Trp His Trp Leu Glu Leu Met Pro Gly Gln Pro Leu Tyr  (SEQ ID        NO:49)                                                                        3.        TGG CAT TGG ATG GAG CTA AGA CCT GGC CAA CCA ATG TAC  (SEQ ID        NO:50)                                                                        encoding  Trp His Trp Met Glu Leu Arg Pro Gly Gln Pro Met Tyr  (SEQ ID        NO:51)                                                                    

Autocrine Mata strain CY599

A MATa strain designed for the expression of peptides in the context ofMFA1 (i.e., using the MFA1 expression vector, Cadus 1239) has beenconstructed. The genotype of this strain, designated CY599, is MATa mfa1mfa2 far1-1 his3::fus1-HIS3 ste2-STE3 ura3 met1 ade1 leu2. In thisstrain, Cadus 1147 (see above) was used to replace STE2 with a hybridgene in which the STE3 coding region is under the control of expressionelements which normally drive the expression of STE2. As a result, thea-factor receptor replaces the α-factor receptor. The genes which encodea-factor are deleted from this strain; the far1 mutation abrogates thearrest of growth which normally follows stimulation of the pheromoneresponse pathway; and the FUS1-HIS3 hybrid gene (integrated at the HIS3locus) provides a selectable signal of activation of the pheromoneresponse pathway. CY599 cells were expected to be capable of thetransport of a-factor or a-factor-like peptides encoded byoligonucleotides expressed from Cadus 1239 by virtue of expression ofthe endogenous yeast transporter, Ste6.

Transport of peptides by the yeast a-factor pathway

Experiments were performed to test: 1. the ability of Cadus 1239 tofunction as a vector for the expression of peptides encoded by syntheticoligonucleotides; 2. the suitability of the oligonucleotides, asdesigned, to direct the export of farnesylated, carboxymethylatedpeptides through the pathway normally used by a-factor; 3. the capacityof CY599 to export these peptides; and 4. the ability of CY599 torespond to those peptides that stimulate the pheromone response pathwayby growing on selective media. These tests were performed using anoligonucleotide which encodes the 12 amino acid a-factor; specifically,the degenerate sequence (NNN)_(n) in the oligonucleotide cloned intoCadus 1239 (see above) (with n=12) encodes the peptide component ofa-factor pheromone. CY599 was transformed with the resulting plasmid(Cadus 1220), and transformants selected on uracil-deficient medium weretransferred to histidine-deficient medium supplemented with a range ofconcentrations of aminotriazole. The results, shown in FIG. 8,demonstrate that the synthetic oligonucleotide, expressed in the contextof MFA1 by Cadus 1220, conferred upon CY599 enhancedaminotriazole-resistant growth on histidine-deficient media. Insummation, these data indicate: 1. Cadus 1220 and the designedoligonucleotide are competent to direct the expression and export of afarnesylated, carboxymethylated peptide encoded by the (NNN)_(n)sequence of the synthetic oligonucleotide; and 2. CY599 can, in anautocrine fashion, respond to a farnesylated, carboxymethylated peptidethat stimulates its pheromone response pathway, in this case signalinginitiates as a-factor binds to STE3.

EXAMPLE 5

Proof of Concept

This example will demonstrate the utility of the autocrine system forthe discovery of peptides which behave as functional pheromoneanalogues. By analogy, this system can be used to discover peptides thatproductively interact with any pheromone receptor surrogates.

CY588 (see strain 5, Example 2 above) will be transformed with CADUS1215 containing oligonucleotides encoding random tridecapeptides for theisolation of functional α-factor analogues (FIG. 4). CY599 (see strain6, Example 2 above) will be transformed with CADUS 1239 containingoligos of random sequence for the isolation of functional a-factoranalogues (FIG. 6). Colonies of either strain which can grow onhistidine-deficient media following transformation will be expanded forthe preparation of plasmid DNA, and the oligonucleotide cloned into theexpression plasmid will be sequenced to determine the amino acidsequence of the peptide which presumably activates the pheromonereceptor. This plasmid will then be transfected into an isogenic strainto confirm its ability to encode a peptide which activates the pheromonereceptor. Successful completion of these experiments will demonstratethe potential of the system for the discovery of peptides which canactivate membrane receptors coupled to the pheromone response pathway.

Random oligonucleotides to be expressed by the expression plasmid CADUS1215 will encode tridecapeptides constructed as

                           (SEQ ID NO:6)                                          5' CGTGAAGCTTAAGCGTGAGGCAGAAGCT(NNK).sub.13 TGATCATCCG,                   

where N is any nucleotide, K is either T or G at a ratio of 40:60 (seeProc Natl Acad Sci 87:6378, 1990; ibid 89:5393, 1992), and the AflII andBclI sites are underlined. This oligonucleotide is designed such that:the AflII and BclI sites permit inserting the oligos into the AflII andBglII site of CADUS 1215 (see FIG. 4); the HindIII site just 5' to theAflII site in the 5' end of the oligo allows future flexibility withcloning of the oligos; the virtual repeat of GAGGCT and the GAGA repeatswhich are present in the wild-type sequence and which can form triplehelixes are changed without altering the encoded amino acids. The randomoligonucleotides described above will actually be constructed from thefollowing two oligos:

    5' CGTGAAGCTTAAGCGTGAGGCAGAAGCT                                                                     (SEQ ID NO:26)                                          and                                                                           5' CGGATGATCA(MNN).sub.13 AGCTTCTG                                                                  (SEQ ID NO:27),                                     

where M is either A or C at a ratio of 40:60. The oligos will beannealed with one another and repetitively filled in, denatured, andreannealed (Kay et al, Gene, 1993). The double-stranded product will becut with AflII and BclI and ligated into the AflII-and BglII-digestedCADUS 1215. The BglII/BclI joint will create a TGA stop codon fortermination of translation of the randomers (FIG. 4). Because of the TAcontent of the Afl overhang, the oligos will be ligated to the AflII-andBglII-digested pADC-MFα at 4° C.

Random oligonucleotides to be expressed by the expression plasmid CADUS1239 will encode monodecapeptides constructed as

    5' GGTACTCGAGTGAAAAGAAGGACAAC(NNK).sub.11 TGTGTTATTGCTTAAGTACG                                                  (SEQ ID NO:12),                         

where N is any nucleotide, K is either T or G at a ratio of 40:60 (seeProc Natl Acad set 87:6378, 1990; ibid 89:5393, 1992), and the XhoI andAflII sites are underlined. When cloned into the XhoI and AflII sites ofCADUS 1239 the propeptides expressed under the control of the ADH1promoter will contain the entire leader peptide of MFal, followed by 11random amino acids, followed by triplets encoding CVIA (the C-terminaltetrapeptide of wild-type a-factor). Processing of the propeptide shouldresult in the secretion of dodecapeptides which contain 11 random aminoacids followed by a C-terminal, farnesylated, carboxymethylatedcysteine.

Using the procedure described above, the oligonucleotides for expressionin CADUS 1239 will actually be constructed from the following twooligos:

                          (SEQ ID NO:28)                                          5' GGTACTCGAGTGAAAAGAAGGACAAC                                                 and                                                                                                 (SEQ ID NO:29)                                          5' CGTACTTAAGCAATAACAca(MNN).sub.11 GTTGTCC,                              

where M is either A or C at a ratio of 40:60, and the XhoI and AflIIsites are underlined.

Discovery of a-factor analogues from a random peptide library

An optimized version of strain 6 (Example 2 above) was derived. Thisyeast strain, CY2012 (MATa ste2-STE3 far1Δ1442 mfa1::LEU2 mfa2-lacZfus1-HIS3 tbt1-1 ura3 leu2 his3 trp1 suc2), was constructed as follows.From a cross of CY570 (MATa mfa1::LEU2 mfa2-lacZ ura3 trp1 his3Δ200 can1leu2 fus1-HIS3 [MFA1 URA3 2μ] [FUS1Δ8-73 TRP1 CEN6]) by CY1624 (MATαtbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 lys2-801 SUC+), a spore wasselected (CY1877) of the following genotype: MATa mfa1::LEU2 mfa2-lacZfus1-HIS3 tbt1-1 ura3 leu2 his3 trp1 suc2. This strain lacks both genes(MFA1 and MFA2) encoding a-factor precursors, contains the appropriatepheromone pathway reporter gene (fus1-HIS3), and transforms byelectroporation at high efficiency (tbt1-1). This strain was altered bydeletion of the FAR1 gene (with Cadus 1442; see Example 6), andreplacement of STE2 coding sequences with that of STE3 (see Example 1)to yield CY2012.

This strain was transformed with plasmid DNA from a random a-factorlibrary by electroporation and plated on 17 synthetic complete plateslacking uracil (-Ura), yielding approximately 10⁵ Ura⁺ colonies perplate after 2 days at 30° C. These colonies were replica plated tohistidine-deficient synthetic complete media (-His) containing 0.2 mM3-aminotriazole and after three days at 30° C. 35 His⁺ replicas werestreaked to -Ura plates. The resultant colonies, 3 from each isolate,were retested for their His⁺ phenotype, and streaked to 5-fluorooroticacid plates to obtain Ura⁻ segregants (lacking a library plasmid). ThoseUra⁻ segregants were tested for the loss of their His⁺ phenotype. Ten ofthe original isolates passed these tests; in two cases only one of thethree Ura⁺ colonies purified from the isolate retained the His⁺phenotype, but nevertheless subsequently segregated Ura⁻ His⁻ colonies.

A single plasmid (corresponding to a bacterial colony) was obtained fromeach of the ten isolates, and reintroduced into CY2012. Eight of the tenplasmids passed the test of retaining the ability to confer the His⁺phenotype on CY2012 (the two that failed correspond to the two isolatesthat were mentioned above, suggesting that these isolates contain atleast one "irrelevant" plasmid). Sequencing of the randomized insert inthe eight plasmids of interest revealed that four contain the sequence:

                          (SEQ ID NO:52)                                          TAT GCT CTG TTT GTT CAT TTT TTT GAT ATT CCG                                                         (SEQ ID NO:53)                                          Tyr Ala Leu Phe Val His Phe Phe Asp Ile Pro,                              

two contain the sequence

                          (SEQ ID NO:54)                                          TTT AAG GGT CAG GTG CGT TTT GTG GTT CTT GCT                                                         (SEQ ID NO:55)                                          Phe Lys Gly Gln Val Arg Phe Val Val Leu Ala,                              

and two contain the sequence

                          (SEQ ID NO:56)                                          CTT ATG TCT CCG TCT TTT TTT TTT TTG CCT GCG                                                         (SEQ ID NO:57)                                          Leu Met Ser Pro Ser Phe Phe Phe Leu Pro Ala                               

Clearly, these sequences encode novel peptides, as the native a-factorsequence differs considerably:

    Tyr Ile Ile Lys Gly Val Phe Trp Asp Pro Ala.                              

The a-factor variants identified from random peptide libraries haveutility as "improved" substrates of ABC transporters expressed in yeast.For example, identification of a preferred substrate of human MDR, onethat retains agonist activity on the pheromone receptor, would permitthe establishment of robust yeast screens to be used in the discovery ofcompounds that affect transporter function.

EXAMPLE 6

Drug screens designed to permit discovery of molecules which modulatethe function of ATP-dependent transmembrane transporters

The availability of cloned DNA encoding the related proteins human Mdr1,human CFTR and human MRP, will allow the construction of yeast strainsexpressing these molecules. The resultant strains will be essential tothe design of microbiological assays which can be used to probe thefunction of these proteins and to discover molecules capable ofinhibiting or enhancing their function in cellular resistance tochemotherapeutics or in ion transport. The present assay makes use ofthe transport of the yeast mating pheromone a-factor from autocrineyeast expressing a human protein capable of substituting for yeast Ste6.

A. MFa1- and Mdr1-containing plasmids obtained from Karl Kuchler(University of Vienna) for use in these experiments

(1) pYMA177 (denoted Cadus 1067), see FIG. 9.

(2) pKK1 includes sequence encoding MFa1 in YEp351; a-factor isoverexpressed from this plasmid due to increased plasmid copy number

(3) pHaMDR1(wt) provides wild type Mdr1 cDNA in a retroviral vector(initially obtained from Michael Gottesman, NIH).

B. Plasmids constructed at Cadus for use in these experiments

A 1.5 kb BamHI-BglII fragment which includes MFa1 sequence was derivedfrom pYMA177 and ligated to BamHI-digested pYMA177 to yield Cadus 1079.Cadus 1079 was digested with BglII and recircularized to delete a 950 bpfragment containing sequence encoding the G185V mutant of human Mdr1;the resultant plasmid is Cadus 1093. pHaMDR1 was digested with BglII toallow isolation of a 965 bp fragment containing wild type human Mdr1(G185) sequence. The 965 bp BglII fragment was inserted intoBglII-digested Cadus 1093 to yield Cadus 1097. The Cadus 1097 constructwas verified by sequencing using dideoxy nucleotides. To yield Cadus1164, pYMA177 was digested with BamHI and recircularized to eliminatethe sequence encoding MFa1. Cadus 1165 was constructed by ligating a 700bp BglII-BamHI fragment from pYMA177 to the large BamHI to BglIIfragment of pYMA177. This results in the removal of both the 1.6 kb MFaBamHI fragment and the 965 bp BglII fragment encoding human Mdr1(G185V); the resulting plasmid is Cadus 1165.

Cadus 1176 resulted from the ligation of a 965 bp BglII fragment frompHaMDR1, containing sequence encoding wild type human Mdr1, toBglII-digested Cadus 1165.

pRS426 (Cadus 1019) served as a URA3 control plasmid.

The final plasmid array used in these experiments is as follows:

    ______________________________________                                                 no       mutant Mdr1                                                                             wt Mdr1                                                    Mdr1     (G185V)   (G185)                                            ______________________________________                                        a-factor   1065       1079      1097                                          overexpression                                                                no a-factor                                                                              1019       1164      1176                                          overexpression                                                                ______________________________________                                    

Evidence for the expression of human Mdr1 constructs in yeast

CADUS plasmids 1065, 1079 and 1097 as well as a URA3 control plasmid(pRS426=1019) were transformed into a ura3⁻, ste6⁻ strain of yeast(WKK6=CY20=MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100ste6::HIS3, obtained from Karl Kuchler). Individual transformants weregrown overnight in SD-URA media and lawns containing approximately 5×10⁶cells were poured onto YPD plates in 3 ml of YPD top agar. Sterilefilter disks were placed on the plates after the top agar hadsolidified, and 5 ml of DMSO or 5 mM valinomycin in DMSO were spottedonto the filter disks. By this assay, expression of mutant Mdr1 fromplasmid 1079 in WKK6 cells conferred weak resistance to valinomycinwhile expression of wild type Mdr1 from plasmid 1097 conferred completeresistance.

Attempts to assay Mdr1 activity by a-factor transport in a two cellsystem

Several attempts to demonstrate mating of WKK6 transformed with thevarious human Mdr1 constructs failed. This is in contrast to publishedexperiments utilizing the mouse mdr3 gene, in which a partialcomplementation of mating deficiency was seen (Raymond et al., 1992).

A "halo" assay was performed by patching WKK6 cells containing thevarious Mdr1 constructs (1019, 1065, 1079 & 1097) onto a lawn of Matαcells which are supersensitive to a-factor (CY32=MATα leu2-3,112trp1-289 ura3-52 his3*1 sst2*2 GAL+) . Although all halos were muchsmaller than those produced by STE6⁺ cells, a small difference in halosize was observed with the relative order 1097>1079˜1065>1019. Thisindicates that a-factor can be transported by the wild-type human Mdr1protein expressed in yeast deleted for STE6, however, the halo assaydoes not appear to be amenable to rapid drug screening.

The halo assay detects a-factor secreted from ste6⁻ Mdr1 strains bygrowth arrest of cells present in the indicator lawn. An alternative,and potentially more sensitive, method makes use of the transcriptionalresponse to pheromone signaling. A strain with an a-factor responsiveHIS3 gene (CY104=MATα ura3 leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1)was cross-streaked with STE6⁺ (CY19=W303-1a=MATa ura3-1 leu2-3,112his3-11,15 trp1-1 ade2-1 can1-100) or ste6⁻ (WKK6=CY20=MATa ura3-1leu2-3,112 his3-11,15 trp1-1 ade2-1 can1-100 ste6::HIS3) strainscontaining various plasmids. The cross-streaking was performed on aplate lacking histidine and tryptophan so that only the CY104 indicatorstrain would grow if stimulated by a-factor. The order of a-factorsecretion seen by this method was CY58>CY61≈CY62≈CY63>CY60 where:CY58=CY19 (STE6⁺, 1019), CY60=CY20 (ste6⁻, 1019), CY61=CY20 (ste6⁻,1065), CY62=CY20 (ste6⁻, 1079) and CY63=CY20 (ste6⁻, 1097). Thus, thedifference in a-factor production between STE6⁺ and ste6⁻ and betweena-factor overproduction or not is detectable in this system, whereas theactivity of Mdr1 in secreting a-factor is not. It was not clear fromthese experiments whether the signal generated from the ste6⁻, a-factoroverproducing strains was due to an alternate pathway for a-factorsecretion or the release of a-factor from lysed cells.

Assay of Mdr1 activity by a-factor transport in an autocrine system

A single strain system for the detection of Mdr1-mediated a-factortransport was constructed in order to improve sensitivity andreproducibility and to circumvent the potential false signal of a-factorrelease from lysed cells (a cell concentration-dependent phenomenon) Astrain was constructed (CY293=MATa ura3 leu2 trp1 his3 fus1-HIS3 can1ade2-1 ste2-STE3 ste6::TRP1) which could respond to a-factor by growingon media lacking histidine and in which the only impediment to thesecretion of a-factor is the lack of a functional STE6 gene. Indeed, theimmediate precursor to this strain, which did contain a functional STE6gene, was able to grow vigorously on -HIS media containing 3 mMaminotriazole, whereas CY293 did not grow at all. The addition ofaminotriazole, a competitive inhibitor of the HIS3 enzyme, is necessaryto reduce the background growth of these strains.

The Mdr1-containing plasmids were introduced into CY293, and thetransformants were streaked onto -HIS plates containing either 0.1 or0.33 mm aminotriazole. The growth pattern thus generated was1097>1067>1065≈1176>1164≈1019. The STE6⁺ parent of CY293 exhibited amuch more vigorous growth than any of these transformants. However,human Mdr1-mediated a-factor secretion is clearly detectable in thisautocrine system. CY293 transformed with 1097 can be used to screen fordrugs which enhance the activity of the Mdr1 protein (increased growthon -HIS+aminotriazole) as well as drugs which inhibit Mdr1 activity(decreased growth on -HIS+aminotriazole). In the latter case, controlsmust be designed to identify compounds which inhibit yeast growth in anMdr1-independent fashion.

Additional strains, with improvements over CY293 (see example 6 [76-35])for the expression of mammalian ABC transporters were constructed. Thesestrains contain the tbt1-1 allele, conferring high-efficiencytransformation by electroporation, and a lesion in the FAR1 gene. Inaddition, they are auxotrophic for tryptophan, and hence can serve ashosts for TRP1-based plasmids.

Two starting strains were selected, CY1555 (MATa tbt1-1 fus1-HIS3 trp1ura3 leu2 his3 lys2-801 SUC+) and CY1557 (MATa tbt1-1 fus1-HIS3 trp1ura3 leu2 his3 suc2) A plasmid containing an internal deletion of FAR1was constructed by amplifying genomic sequences corresponding to the 5'end and the 3' end of FAR1, and ligating them together in pRS406 (anintegrative vector containing the URA3 gene), thereby creating adeletion from the 50th to the 696th predicted codon of FAR1.

The oligonucleotides used for amplification were: for the 5' segment ofFAR1:

                          (SEQ ID NO:58)                                          5'-CGGGACCGATGCAATTTTCAACATGC-3'   (23FAR1)                                   and                                                                                                 (SEQ ID NO:59)                                          5'-GCTCTAGATGCTACTGATCCCGC-3'      (1RAF616)                              

and for the 3' segment of FAR1:

                          (SEQ ID NO:60)                                          5'-CGCCGCATGACTCCATTG-3'          (2552FAR1)                                  and                                                                                                 (SEQ ID NO:61)                                          5'-GGGGTACCAATAGGTTCTTTCTTAGG-3'  (1RAF2979).                             

The resultant amplification products were restricted with BamHI and XbaI(5' segment; 0.6 kb) or NheI and KpnI (3' segment; 0.4 kb), and ligatedinto pRS406 (Cadus 1011) that had been restricted with BamHI and KpnI.The resultant plasmid (Cadus 1442) was restricted with EcoRI to directintegration at the FAR1 locus, Ura+ transformants were purified andsubjected to selection on 5-fluoro-orotic acid, and Ura- clones werescreened for the impaired mating ability conferred by the far1 deletion.The STE6 gene was subsequently deleted using the ste6::hisG-URA3 plasmid(Cadus 1170; constructed by Karl Kuchler), and the STE2 coding sequenceswere replaced with STE3 coding sequences as described in Example 1. Theresultant strains were named CY1880 (MATa ste2-STE3 ste6::hisG far1Δ1442tbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 suc2) and CY1882 (MATa ste2-STE3ste6::hisG far1Δ1442 tbt1-1 fus1-HIS3 trp1 ura3 leu2 his3 lys2-801SUC+).

Like strain CY293 these strains show a dramatic enhancement of growth(via the fus1-HIS3 reporter gene) under conditions of histidinestarvation when transformed with the MDR1-encoding plasmids Cadus 1097and Cadus 1176 (as compared to control plasmids lacking MDR1, Cadus 1065and 1019) In addition, these strains display more robust general growth,transform by electroporation at high efficiency (the tbt1-1 effect),lack susceptibility to pheromone-induced growth arrest (due toinactivation of FAR1), and act as hosts for TRP1-based plasmids(ste6::hisG instead of ste6::TRP1).

These strains also act as suitable hosts for the STE6-CFTR chimerasconstructed by Teem et al. (1993) (see Example 7). When compared withCY293 in their ability to distinguish between wild type and ΔF508STE6-CFTR in their ability to transport a-factor, a similar enhancementis seen:

    ______________________________________                                        Host strain                                                                              wild type/ΔF508 growth ratio                                 ______________________________________                                        CY293      18                                                                 CY1880     7                                                                  CY1882     8                                                                  ______________________________________                                    

The strains were transformed with Cadus 1515 (STE6-CFTR(H5) URA3 CEN) or1516 (STE6-CFTR(H5)ΔF508 URA3 CEN) and inoculated at various celldensities (OD₆₀₀ =0.003 to 0.048) into histidine-free media containingvarious concentrations of 3-aminotriazole (0 to 1.2 mM). After overnightgrowth in microtiter wells the optical densities at 600 nm of the wellswere measured, and ratios for wild type vs. ΔF508 calculated. The ratiosreflect the highest ratios obtained in this experiment but notnecessarily the highest ratio it would be possible to obtain.

Improvements on the autocrine yeast expressing Mdr1

The results described above indicate that the human Mdr1 proteintransports a-factor less efficiently than does the yeast STE6 protein.Attempts will be made to isolate mutant a-factor molecules which aretransported more efficiently by human Mdr1 and yet which retain agonistactivity on the a-factor receptor (STE3 protein). To do this, a-factorcoding sequences will be chemically synthesized using "dirty"nucleotides and inserted into an a-factor expression cassette. Anexample of "dirty" synthesis would be to incorporate nucleotide from amixture of 70% G and 10% each of A, T and C at a position in thea-factor sequence where G would normally appear. Using oligonucleotidesgenerated in this manner, a diverse library of peptides can be expressedin yeast and screened to identify those peptides which retain theability to signal to the STE3 protein but which are also a favorablesubstrate for transport by human Mdr1.

A second improvement to the system will be the addition of apheromone-inducible "negative selection" marker. For instance, the FUS1promoter can be connected to GAL1 coding sequences. Expression of GAL1is toxic in the presence of galactose in strains which contain mutationsin either the GAL7 or GAL10 genes. In the context of an autocrine Mdr1strain, this selection system should render cells galactose-sensitive.Addition of a compound which inhibits the ability of the Mdr1 protein tosecrete a-factor would allow this strain to grow on galactose-containingmedia. This selection system would also eliminate false positives due tolethality. Controls must still be designed to identify compounds whichinterfere at other points in the pheromone response pathway.

The third improvement in the system involves inactivation of yeast geneswhich function equivalently to mammalian MDR genes. A network of genesinvolved in pleiotropic drug resistance (PDR) have been identified inyeast. This modification will be useful in any yeast screen designed toassay the interaction of compounds with intracellular targets. Theimproved autocrine yeast strain, expressing human Mdr1, will be used toscreen compound libraries for molecules which inhibit the transportfunction of this protein. In addition, Mfα expression cassettescontaining oligonucleotides encoding random peptides will be expressedin the autocrine Mdr1 strain to identify peptides capable of inhibitingthe transport of a-factor, or an a-factor analogue by Mdr1.

EXAMPLE 7

Identification of an analogue of yeast a-factor that is transported bywild type human CFTR

This example describes the use of autocrine yeast strains to identifymolecules capable of enhancing transport by dysfunctional ATP-dependenttransmembrane transporters, e.g. mutant human CFTR proteins. The wildtype human CFTR protein will not substitute for Ste6 function in yeastby transporting native a-factor pheromone (John Teem, unpublishedobservations). In order to maximally exploit autocrine yeast strains forthe discovery of molecules which enhance mutant CFTR function, ana-factor-like peptide which can serve as a substrate for transport byCFTR will be identified. An a-factor analogue must also bindfunctionally to the pheromone receptor, Ste3, in order to initiatepheromone signalling. The CFTR transport substrate will be identified byexpressing a randomly mutated MFa expression cassette in autocrine yeastdeleted for Ste6 expression, but expressing the wild type human CFTRprotein.

Expression of mutant human CFTR proteins in autocrine yeast

Transport of an a-factor-like peptide pheromone by CFTR will serve asthe basis for the design of screens to be used in the identification ofcompounds which augment the transport function of CFTR proteinscontaining cystic fibrosis (CF) mutations. Based on studies done by Teemet al. (1993) using Ste6/CFTR chimeras, mutant CFTR is not expected toefficiently transport an a-factor analogue. Teem et al. (1993) have madechimeric proteins by substituting varying portions of the sequencesencoding the first nucleotide binding domain of human CFTR for analogoussequence in yeast STE6. The chimeric proteins will transport nativeyeast a-factor when expressed in yeast. However, introduction of a CFmutation (ΔF508) reduces the ability of these proteins to transport theyeast pheromone. Teem et al. have also identified revertants in yeast,i.e., proteins containing second site mutations which restore theability of chimeras bearing the CF mutation to transport a-factor.Introduction of the revertant mutations into defective CFTR proteinexpressed in mammalian cells decreased in part the processing andchannel defects of the ΔF508 protein.

With the identification of an analogue of yeast a-factor that will serveas a substrate for transport by CFTR, mutations can be introduceddirectly into the human CFTR protein expressed in yeast, eliminating thenecessity of creating chimeric proteins containing Ste6 sequence. Thiswill permit the targeting of potential CF therapeutics to the entirehuman CFTR molecule.

Mutations of interest include G551D, N1303K and ΔI507 in addition toΔF508; these mutations are naturally occurring, give rise to cysticfibrosis in affected individuals, and appear to affect either thetransport and processing of CFTR or the regulation of function of thatprotein (Welsh and Smith 1993). These mutations also rank among the mostcommon alterations of CFTR thus far identified in CF patients.

If a peptide which will serve as a substrate for transport by CFTRcannot be identified, chimeric Ste6/CFTR proteins and unaltered a-factorcan be utilized in screens based on autocrine strains of yeast. Thesestrains offer distinct advantages in screening applications: easyadaptability to large-scale automation, simplicity and increasedsensitivity when compared to traditional yeast mating cell assays ofpheromone signaling, and the potential to employ the instant technologyto identify active peptide structures.

Identification of compounds that enhance transport of a-factor-likepeptide by mutant CFTR

Autocrine strains expressing mutant human CFTR protein will be capableof growth on rich media but, due to inadequate transport of andsignaling by an a-factor analogue, will not grow efficiently onselective (histidine-deficient) media. Pheromone signaling in thesestrains initiates expression from a FUS1 promoter sequence controllingtranscription of the His3 enzyme; expression of His3 is required forgrowth on media lacking histidine. These strains will be used to screencompound libraries to identify molecules which reverse the inability ofthe mutant CFTR to transport a-factor analogue and permit growth of theyeast on histidine-deficient medium. Alternatively, active compoundswould be capable of signaling to the a-factor receptor, Ste3, directly,or may interact with the pheromone response pathway elsewhere. Suitablecontrols would differentiate among these possibilities.

Identification of random peptides that enhance transport by mutant CFTR

Plasmids containing oligonucleotides which encode random peptides willbe expressed in autocrine yeast bearing a mutant human CFTR. Thesepeptides, expressed using α-factor-based expression cassettes, will betransported to the extracellular environment via the yeast secretorypathway. Peptides of interest will be identified by virtue of theirability to permit the growth of a mutant CFTR-containing strain onhistidine-deficient medium. Active peptides would permit transport ofa-factor analogue by the mutant human CFTR. Alternatively, a peptide mayinteract at other points along the pheromone response pathway. Suitablecontrols would differentiate between these possible outcomes.

EXAMPLE 8

Prophetic Example of Substitution of Prohormone Convertase PC1 for YeastKEX2

The mammalian prohormone convertases PC1/PC3 and PC2 are involved in theproteolytic processing of proopiomelanocortin (POMC), with PC1preferentially releasing adrenocorticotropic hormone (ACTH) andβ-lipotropin and PC2 preferentially releasing β-endorphin, N-terminallyextended ACTH containing the joining peptide (JP), and eitherα-melanocyte-stimulating hormone (α-MSH) or des-acetyl α-MSH (Benjannet,S., et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88:3564-3568; Seideh N.G. et al. (1992) FEBS Lett. 310, 235-239). By way of example, a yeaststrain is described in which mammalian PC1 processes a chimericpre-pro-POMC/α-factor peptide, permitting the secretion of matureα-factor and stimulation of the screening strain in an autocrine fashionto histidine protrotrophy. Autocrine strains will be constructed inwhich: 1. yeast KEX2 is disrupted; 2. yeast KEX2 activity is substitutedwith that of mammalian PC1; 3. a novel MFα construct containing thedibasic cleavage site recognized by PC1 (in place of the KEX2recognition sequence) will be expressed; 4. production of matureα-factor will require PC1 activity; and 5. growth of the strain will bestimulated by the production of mature α-factor.

The genotype of the parental strain for this example is MATa bar1::hisGfar1-1 fus1-HIS3 ste14::TRP1 ura3 trp1 leu2 his3. Initially, the KEX2allele of this strain will be disrupted using an integrating plasmid(pkex2Δ) encoding the flanking regions of the KEX2 locus with the entirecoding regions from the initiator codon to the terminator codon deleted.Cleavage of pkex2Δ₋₋ with Bsu36I followed by transfection into theparental strain will result in integration of this plasmid into the KEX2locus. Transformation can be scored as conversion to uracil prototrophy.Subsequent transfer of URA+ transformants to plates containing5-fluoroorotic acid results in the growth of colonies with the kex1Δ₁₃allele. Integration can be confirmed by Southern blot analysis and-bycolony PCR using oligonucleotide primers flanking the KEX2 locus. Thiskex1Δ₋₋ strain will be able to grow on histidine-deficient media in thepresence of exogenenously added α-factor, but will not be able to growin an autocrine fashion on histidine-deficient media once transfectedwith Cadus 1219 (URA3 2mu-ori REP3 AmpR f1-ori α-factor) sinceprocessing of the pre-pro-POMC/α-factor chimera expressed from Cadus1219 requires KEX2 activity.

In the screening strain, PC1 activity will substitute for the deletedKEX2 activity in the maturation of α-factor peptide. PC1 cDNA (accession# M69196) obtained from mouse (Korner et al (1991) Proc. Natl. Acad. SciU.S.A. 88:6834-6838) was found to encode a protein of 753 amino acids.Sequences encoding PC1 will be cloned into a high copy replicatingplasmid (Cadus 1289). Yeast cells transformed with this plasmid willacquire the ability to grow on leucine-deficient media and will expresshigh levels of PC1 protein due to the presence of the PGK promotor.

A hybrid gene encoding the prepro-region of human POMC (accession #K02406; Takahashi, H., et al (1983) Nucleic Acids Research 11:6847-6858)and the coding region of a single repeat of mature α-factor will beconstructed in the following fashion. The prepro-region of human POMCwill be amplified with an HindIII site at the 5' end and a BbsI site atthe 3' end using VENT polymerase and the following primers: 5'GGGAAGCTTATGCCGAGATCGTGCTGCCAGCCGC 3' (SEQ ID NO:30) (HindIII site isunderlined and initiation codon is italic bold) and antisense 5'GGGGAAGACTTCTGCCCTGCGCCGCTGCTGCC 3' (SEQ ID NO:31) (BbsI recognition isunderlined), leaving the amino acid sequence -SSGAGQKR- at the 3' endwith a Bbs1 site leaving an overhang at the -KR- dibasic cleavagesequence. The coding region of α-factor will be amplified from Cadus1219 with a Bbs1 site at the 5' end and a BglII site at the 3' end usingthe primers 5' GGGGAAGACCCGCAGGAGGCAGAAGCTTGGTTGCAG 3' (SEQ ID NO:32)(BbsI site is underlined) and 5' GGGAGATCTTCAGTACATTGGTTGGCC 3' (SEQ IDNO:33) (BglII site is underlined, termination codon is bold). The PCRfragment encoding the pre-pro segment of POMC is restricted with HindIIIand BbsI and gel purifed, the PCR fragment encoding₋₋ α-factor is cutwith BbsI and BglII and gel purified, and Cadus 1215 is cut with BglIIand partially with HindIII and the HindIII-BglII restricted vectorcontaining the pAlter polylinker sequences is gel purified. Three-partligation of the two PCR products with HindIII and BglII digested Cadus1215 will yield a hybrid POMC/α-factor gene in which the first 104 aminoacids residues are from POMC and the remaining 17 are from α-factor. Thestructure of this hybrid gene around the PC1 cleavage site is:--RNSSSSGSSGAGOKREAEAWHWLQLKPGQPMY* (SEQ ID NO:34) where residuesdonated by POMC are underlined, the dibasic cleavage site is underlinedbold, and the sequence of mature α-factor is in italics. Thetetrapeptide -EAEA- juxtaposed between the dibasic cleavage site and theamino-terminal tryptophan of mature α-factor should be removed by thedipeptidyl aminopeptidase activity of ste13p.

Introduction of the PC1-encoding plasmid and the POMC/α-factor plasmidinto a kex2Δ₋₋ strain with the genotype MATa bar1::hisG far1-1 fus1-HIS3ste14::TRP1 ura3 trp1 leu2 his3 should result in autocrine growth thatis dependent on PC1-mediated cleavage of the dibasic motif at theamino-terminal side of the single copy of α-factor encoded by thePOMC/α-factor plasmid. That is, the autocrine behaviour of this strainshould be due to expression of PC1. A suitable negative control isprovided by expression of mammalian PC2 in place of PC1; the cleavagesite of POMC that is inserted upstream of the₋₋ α-factor gene is notrecognized by PC2. Therefore, it should be possible to construct strainsspecific for PC1- versus PC2-dependent processing by judicious choice ofcleavage sites from POMC to append to the 5' end of the α-factor gene.Compounds or random peptides that disrupt the autocrine growth of thisstrain but which do not have growth inhibitory effects when this strainis grown in histidine-containing media are potential inhibitors of PC1activity.

EXAMPLE 9

Prophetic Example of Substitution of Human MEK (MAP Kinase Kinase) forYeast STE7

In order to develop a screen for compounds which act as inhibitors of amammalian kinase, one could construct a yeast strain that is deficientin STE7 activity, and which contains human MEK in a yeast expressionvector. In addition, the strain would be equipped with reportercapacities, as described below.

To disrupt the yeast STE7 gene, the following approach could be taken:pBluescriptKS+, a multicopy E. coli vector, is engineered to containSTE7 sequences deleted for 5' noncoding and promoter sequence and for asizeable portion of the coding region. The |ste7 sequence is thensubcloned into pRS406|ClaI, a Bluescript-based plasmid containing theyeast URA3 gene and the resulting plasmid, pRS406|ClaI|ste7, is used todisrupt the wild type STE7 gene as follows. pRS406|ste7 is digested withClaI, and used to transform yeast strain CY252 (genotype MATaste14::TRP1 fus1-HIS3 far1-1 ura3 leu2 trp1 his3 ade2-1 met1) to uracilprototrophy. Subsequent transfer of Ura+ transformants to mediacontaining 5-fluoroorotic acid results in colonies containing the ste7|allele, which is confirmed by Southern analysis and by the inability ofthe strain to grow in the absence of histidine when stimulated with αpheromone.

To construct a plasmid capable of expressing human MEK in yeast cells,the following oligonucleotides are constructed: 5'CCGCGTCTCACATGCCCAAGAAGAAGCCG-3' (SEQ ID NO:35) (forward) and 5'CCGTCTAGATGCTGGCAGCGTGGG-3' (SEQ ID NO:36) (reverse). When used in apolymerase chain reaction (PCR) with human cDNA as template, theseprimers will direct the amplification of DNA encoding human MEK. Toinsert the human MEK gene into a yeast expression vector, the PCRproduct is digested with Esp3I and XbaI (bold sequences above) andligated to the yeast-E. coli shuttle plasmid Cadus 1289, previouslydigested with NcoI and XbaI. The resulting plasmid will replicateautonomously in yeast cells, confer leucine prototrophy to a yeast leu2mutant, and direct the expression of MEK from the constitutive PGK1promoter.

When the above-described PGK1-MEK plasmid is introduced into theCY252-ste7| cells, it should restore their ability to grow in theabsence of histidine when incubated with α pheromone, due to the abilityof MEK to functionally replace STE7 in the pheromone response pathwayand thereby effect the stimulation of the fus1-HIS3 fusion situated inthe chromosome. One could then screen for compounds which are able toreverse α pheromone-dependent growth in the absence of histidine, butwhich have no nonspecific toxic effect in the presence of histidine.

In an autocrine embodiment, the yeast cells made ste7| are of thegenotype MATa bar1::hisG far1-1 fus1-HIS3 ste14::trp1::LYS2 ura3 trp1leu2 his3 lys2 (CY588trp). The procedure followed is exactly as abovefor CY252. After construction of CY588trpste7|, the cells created aretransformed with the plasmid expressing MEK, as well as with a plasmidcapable of expressing secreted α pheromone. This transformed strain(CY588trpste7|[MEK/MFα]) should be able to grow on media lackinghistidine and containing high (20 22222222222222 mM) amounts of3-aminotriazole. The growth of this strain on this media should bestrictly dependent on the presence of both plasmids (each necessary,neither sufficient). Compounds that interfere with this growth could betested as above, with the exception that exogenously added α pheromoneis unnecessary.

In addition, CY588trpste7|[MEK/MFα] could be transformed with a plasmidlibrary expressing cytoplasmically targeted random peptides. Those thatinterfere with the function of MEK would be identified by replicaplating to media deficient in histidine (and potentially containing3-aminotriazole); cells expressing such inhibitory peptides would beHis⁻. Such a screen could be streamlined with the addition of reporterconstructs with the potential of negative selection, such as fus1-URA3or fus1-GAL1. In this case inhibitory peptides would confer on thetarget strain the ability to grow on 5-FOA- (for cells with diminishedfus1-URA3) or galactose- (for cells with diminished fus1-GAL1 in agal10⁻ background) containing media.

Confirmatory tests would include biochemical assay of the activity ofMEK (either in vitro or in vivo) in the presence of random peptides orother molecules identified as potential MEK inhibitors.

EXAMPLE 10

Functional Expression of a Mammalian G Protein-Coupled Receptor andLigand in an Autocrine Yeast Strain

In this example we describe a set of experiments that detail theaccomplishment of the following: (1) expression of human C5a receptor inyeast; (2) expression of the native ligand of this receptor, human C5a,in yeast; and (3) activation of the endogenous yeast pheromone pathwayupon stimulation of the C5a receptor by C5a when both of these moleculesare expressed within the same strain of autocrine yeast. Following theexperimental data we outline the utility of autocrine strains of yeastthat functionally express the human C5a receptor.

Human C5a is a 74 amino acid polypeptide that derives from the fifthcomponent of complement during activation of the complement cascade; itis the most potent of the complement-derived anaphylatoxins. C5a is apowerful activator of neutrophil and macrophage functions includingproduction of cytotoxic superoxide radicals and induction of chemotaxisand adhesiveness. In addition C5a stimulates smooth muscle contraction,induces degranulation of mast cells, induces serotonin release fromplatelets and increases vascular permeability. The C5a anaphylatoxin canalso amplify the inflammatory response by stimulating the production ofcytokines. As C5a is a highly potent inflammatory agent, it is a primarytarget for the development of antagonists to be used for intervention ina variety of inflammatory processes.

The C5a receptor is present on neutrophils, mast cells, macrophages andsmooth muscle cells and couples through G proteins to transmit signalsinitiated through the binding of C5a.

Expression of the C5a Receptor

The plasmid pCDM8-C5aRc, bearing cDNA sequence encoding the human C5areceptor, was obtained from N. Gerard and C. Gerard (Harvard MedicalSchool, Boston, Mass.) (Gerard and Gerard 1991). Sequence encoding C5awas derived from this plasmid by PCR using VENT polymerase (New EnglandBiolabs Inc., Beverly Mass.), and the following primers:

                          (SEQ ID NO:62)                                          #1-GGTGGGAGGGTGCTCTCTAGAAGGAAGTGTTCACC                                                              (SEQ ID NO:63)                                          #2-GCCCAGGAGACCAGACCATGGACTCCTTCAATTATACCACC.                             

Primer #1 contains a single base-pair mismatch (underlined) to C5areceptor cDNA. It introduces an XbaI site (in bold) 201 bp downstreamfrom the TAG termination codon of the C5a receptor coding sequence.Primer #2 contains two mismatched bases and serves to create an NcoIsite (in bold) surrounding the ATG initiator codon (double underlined)The second amino acid is changed from an aspartic acid to an asparagineresidue. This is the only change in primary amino acid sequence from thewild type human C5a receptor.

The PCR product was restricted with NcoI and XbaI (sites in bold) andcloned into CADUS 1002 (YEp51Nco), a Gal10 promoter expression vector.The sequence of the entire insert was determined by dideoxy sequencingusing multiple primers. The sequence between the NcoI and XbaI sites wasfound to be identical to the human C5a receptor sequence that wasdeposited in GenBank (accession# J05327) with the exception of thosechanges encoded by the PCR primers. The C5a receptor-encoding insert wastransferred to CADUS 1289 (pLPXt), a PGK promoter expression vector,using the NcoI and XbaI sites, to generate the C5a receptor yeastexpression clone, CADUS 1303.

A version of the C5a receptor which contains a yeast invertase signalsequence and a myc epitope tag at its amino terminus was expressed inCadus 1270-transferred yeast under control of a GAL10 promoter. Plasmidsencoding an untagged version of the C5a receptor and a myc-taggedderivative of FUS1 served as controls. The expression of the taggedreceptor in yeast was confirmed by Western blot using the anti-mycmonoclonal antibody 9E10. In the lane containing the extract from theCadus 1270-transformant, the protein that is reactive with the anti-mycmonoclonal antibody 9E10 was approximately 40 kD in size, as expected.Note that this receptor construct is not identical to the one used inthe autocrine activation experiments. That receptor is not tagged, doesnot contain a signal sequence and is driven by the PGK promoter.

Expression of the Ligand, C5a

A synthetic construct of the sequence encoding C5a was obtained from C.Gerard (Harvard Medical School, Boston, Mass.). This synthetic gene hadbeen designed as a FLAG-tagged molecule for secretion from E. coli(Gerard and Gerard 1990). The C5a coding region, still containing E.coli codon bias, was amplified using VENT polymerase (New EnglandBiolabs Inc., Beverly Mass.) through 30 cycles using the followingprimers:

                          (SEQ ID NO:64)                                          C5a5' = CCCCTTAAGCGTGAGGCAGAAGCTACTCTGCAAAAGAAGATC                            and                                                                                                 (SEQ ID NO:65)                                          C5a3' = GAAGATCTTCAGCGGCCGAGTTGCATGTC                                     

A PCR product of 257 bp was gel isolated, restricted with AflII andBglII, and cloned into CADUS 1215 (an expression vector designed toexpress peptide sequences in the context of Mfα) to yield CADUS 1297.The regions of homology to the synthetic C5a gene are underlined. The 5'primer also contains pre-pro α-factor sequence. Upon translation andprocessing of the pre-pro α-factor sequence, authentic human C5a shouldbe secreted by yeast containing CADUS 1297. The insert sequence in CADUS1297 was sequenced in both orientations by the dideoxy method and foundto be identical to that predicted by the PCR primers and the publishedsequence of the synthetic C5a gene (Franke et al. 1988).

Two sets of experiments, aside from the autocrine activation of yeastdetailed below, demonstrated that CADUS 1297 can be used to express C5ain yeast.

1.) C5a was immunologically detected in both culture supernatant andlysed cells using a commercially available enzyme-linked immunosorbentassay (ELISA) (Table 3). This assay indicated the concentration of C5ain the culture supernate to be approximately 50 to 100 nM. Incomparison, in data derived from mammalian cells, the binding constantof C5a to its receptor is 1 nM (Boulay et al. 1991).

2.) C5a expressed in yeast was shown to compete for binding withcommercially obtained (Amersham Corporation, Arlington Heights, Ill.),radiolabeled C5a on induced HL60 cells.

Activation Of the Pheromone Response Pathway In Autocrine YeastExpressing the Human C5a Receptor and Human C5a

Activation of the yeast pheromone response pathway through theinteraction of C5a with the C5a receptor was demonstrated using a growthread-out. The strain used for this analysis, CY455 (MATα tbtl-1 ura3leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1 ste3*1156), contains thefollowing significant modifications. A pheromone inducible HIS3 gene,fus1-HIS3, is integrated at the FUS1 locus. A hybrid gene containingsequence encoding the first 41 amino acids of GPA1 (the yeast Gαsubunit) fused to sequence encoding human Gαi2a (minus codons for theN-terminal 33 amino acids) replaces GPA1 at its normal chromosomallocation. The yeast STE14 gene is disrupted to lower the basal level ofsignaling through the pheromone response pathway. The yeast a-factorreceptor gene, STE3, is deleted. The last two modifications are probablynot essential, but appear to improve the signal-to-noise ratio.

CY455 (MATα tbt1-1 ura3 leu2 trp1 his3 fus1-HIS3 can1 ste14::TRP1ste3*1156) was transformed with the following plasmids:

Cadus 1289+Cadus 1215=Receptor⁻ Ligand⁻ =(R-L-)

Cadus 1303+Cadus 1215=Receptor⁺ Ligand⁻ =(R+L-)

Cadus 1289+Cadus 1297=Receptor⁻ Ligand⁺ =(R-L+)

Cadus 1303+Cadus 1297=Receptor⁺ Ligand⁺ =(R+L+)

Receptor refers to the human C5a receptor.

Ligand refers to human C5a.

Three colonies were picked from each transformation and grown overnightin media lacking leucine and uracil, at pH 6.8 with 25 mM PIPES (LEU⁻URA⁻ pH6.8 with 25 mM PIPES). This media was made by adding 0.45 ml ofsterile 1M KOH and 2.5 ml of sterile 1M PIPES pH 6.8 to 100 ml ofstandard SD LEU⁻ URA⁻ media. After overnight growth the pH of this mediais usually acidified to approximately pH 5.5. Overnight cultures werewashed once with 25 mM PIPES pH 6.8 and resuspended in an equal volumeof media lacking leucine, uracil and histidine (LEU⁻ URA⁻ HIS⁻ pH6.8with 25 mM PIPES). The optical density at 600 nm of a 1/20 dilution ofthese cultures was determined and the cultures were diluted into 25 mMPIPES pH 6.8 to a final OD₆₀₀ of 0.2. A volume (5 μl) of this dilutionequivalent to 10,000 cells was spotted onto selective (HIS⁻ TRP⁻ pH6.8+1mM aminotriazole) or non-selective (HIS⁺ TRP⁻ pH6.8) plates Only thosestrains expressing both C5a and its receptor (R+L+) show growth on theselective plates which lack histidine. All test strains are capable ofgrowth on plates containing histidine. The R+L+ strain will grow onplates containing up to 5 mM aminotriazole, the highest concentrationtested.

For verification of pheromone pathway activation and quantification ofthe stimulation, the activity of the fus1 promoter was determinedcolorometrically using a fus1-lacZ fusion in a similar set of strains.CY878 (MATα tbt1-1 fus1-HIS3 can1 ste14::trp1::LYS2 ste3*1156gpa1(41)-Gαi2) was used as the starting strain for these experiments.This strain is a trp1 derivative of CY455. The transformants for thisexperiment contained CADUS 1584 (pRS424-fus1-lacZ) in addition to thereceptor and ligand plasmids. Four strains were grown overnight in SDLEU⁻ URA⁻ TRP⁻ pH6.8 with 50 mM PIPES to an OD₆₀₀ of less than 0.8.Assay of β-galactosidase activity (Guarente 1983) in these strainsyields the data shown in FIG. 10.

Projected Uses of the Autocrine C5a Strains

A primary use of the autocrine C5a strains will be in the discovery ofC5a antagonists. Inhibitors of the biological function of C5a would beexpected to protect against tissue damage resulting from inflammation ina wide variety of inflammatory disease processes including but notlimited to: respiratory distress syndrome (Duchateau et al. 1984;Hammerschmidt et al. 1980), septic lung injury (Olson et al. 1985),arthritis (Banerjee et al. 1989), ischemic and post-ischemic myocardialinjury (Weisman 1990; Crawford et al. 1988) and burn injury (Gelfand etal. 1982).

The autocrine C5a system as described can be used to isolate C5aantagonists as follows:

1. High throughput screens to identify antagonists of C5a

A straightforward approach involves screening compounds to identifythose which inhibit growth of the R+L+ strain described above inselective media but which do not inhibit the growth of the same strainor of a R+L- strain in non-selective media. The counterscreen isnecessary to eliminate from consideration those compounds which aregenerally toxic to yeast. Initial experiments of this type have led tothe identification of compounds with potential therapeutic utility.

2. Identification of antagonists using negative selection

Replacement of the fus1-HIS3 read-out with one of several negativeselection schemes (fus1-URA3/FOA, fus1-GAL1/galactose or deoxygalactose,FAR1 sst2 or other mutations that render yeast supersensitive for growtharrest) would generate a test system in which the presence of anantagonist would result in the growth of the assay strain. Such anapproach would be applicable to high-throughput screening of compoundsas well as to the selection of antagonists from random peptide librariesexpressed in autocrine yeast. Optimization of screens of this type wouldinvolve screening the R+L+ strain at a concentration of amino-triazolewhich ablates growth of the R+L- strain (we are currently using 0.6 to0.8 mM) and counterscreening the R+L- strain at a concentration ofaminotriazole which gives an identical growth rate (we are using 0.14mM). In addition, the system could employ one of several colorometric,fluorescent or chemiluminescent readouts. Some of the genes which can befused to the fus1 promoter for these alternate read-outs include lacZ(colorometric and fluorescent substrates), glucuronidase (colorometricand fluorescent substrates), phosphatases (e.g. PHO3, PHO5, alkalinephosphatase; colorometric and chemiuminescent substrates), green protein(endogenous fluorescence), horse radish peroxidase (colorometric),luciferase (chemiluminescence).

The autocrine C5a strains have further utility as follows:

3. In the identification of novel C5a agonists from random peptidelibraries expressed in autocrine yeast

Novel peptide agonists would contribute to structure/function analysesused to guide the rational design of C5a antagonists.

4. In the identification of receptor mutants.

Constitutively active, that is, ligand independent, receptors may beselected from highly mutagenized populations by growth on selectivemedia. These constitutively active receptors may have utility inpermitting the mapping of the sites of interaction between the receptorand the G-protein. Identification of those sites may be important to therational design of drugs to block that interaction. In addition,receptors could be selected for an ability to be stimulated by someagonists but not others or to be resistant to antagonist. These variantreceptors would aid in mapping sites of interaction between receptor andagonist or antagonist and would therefore contribute to rational drugdesign efforts.

5. In the identification of molecules that interact with Gαi2

Compounds or peptides which directly inhibit GDP exchange from Gαi2would have the same effect as C5a antagonists in these assays.Additional information would distinguish inhibitors of GDP exchange fromC5a antagonists. This information could be obtained through assays thatdetermine the following:

1. inhibition by test compounds of Gαi2 activation from other receptors,

2. failure of test compounds to compete with radiolabeled C5a forbinding to the C5a receptor,

3. failure of test compounds to inhibit the activation of other Gαsubunits by C5a, and

4. inhibition by test compounds of signalling from constitutively activeversions of C5a, or other, receptors.

EXAMPLE 11

Construction of Hybrid Gα Genes Construction of two sets of chimericyeast/mammalian Gα genes, GPA₄ -Gα and GPA1_(Bam) -Gα

The Gα subunit of heterotrimeric G proteins must interact with both theβγ complex and the receptor. Since the domains of Gα required for eachof these interactions have not been completely defined and since ourfinal goal requires Gα proteins that communicate with a mammalianreceptor on one hand and the yeast βγ subunits on the other, we desiredto derive human-yeast chimeric Gα proteins with an optimized ability toperform both functions. From the studies reported here we determinedthat inclusion of only a small portion of the amino terminus of yeast Gαa is required to couple a mammalian Gα protein to the yeast βγ subunits.It was anticipated that a further benefit to using these limitedchimeras was the preservation of the entire mammalian domain of the Gαprotein believed to be involved in receptor contact and interaction.Thus the likelihood that these chimeras would retain their ability tointeract functionally with a mammalian receptor expressed in the sameyeast cell was expected to be quite high.

Plasmid constructions

pRS416-GPA1 (Cadus 1069). An XbaI-SacI fragment encoding the entire GPA1promotor region, coding region and approximately 250 nucleotides of 3'untranslated region was excised from YCplac111-GPA1 (from S. Reed,Scripps Institute) and cloned into YEp vector pRS416 (Sikorski andHieter, Genetics 122: 19 (1989)) cut with XbaI and SacI.

Site-directed mutagenesis of GPA1 (Cadus 1075, 1121 and 1122). A 1.9 kbEcoRI fragment containing the entire GPA1 coding region and 200nucleotides from the 5' untranslated region was cloned into EcoRI cut,phosphatase-treated pALTER-1 (Promega) and transformed byelectroporation (Biorad Gene Pulser) into DH5αF' bacteria to yield Cadus1075. Recombinant phagemids were rescued with M13KO7 helper phage andsingle stranded recombinant DNA was extracted and purified according tothe manufacturer's specifications. A new NcoI site was introduced at theinitiator methionine of GPA1 by oligonucleotide directed mutagenesisusing the synthetic oligonucleotide:

                          (SEQ ID NO:66)                                          5' GATATATTAAGGTAGGAAACCATGGGGTGTACAGTGAG 3'.                             

Positive clones were selected in ampicillin and several independentclones were sequenced in both directions across the new NcoI site at +1.Two clones containing the correct sequences were retained as Cadus 1121and 1122.

Construction of a GPA1-based expression vector (Cadus 1127). The vectorused for expression of full length and hybrid mammalian Gα proteins inyeast, Cadus 1127, was constructed in the following manner. A 350nucleotide fragment spanning the 3' untranslated region of GPA1 wasamplified with Taq polymerase (AmpliTaq; Perkin Elmer) using theoligonucleotide primers A

    (5' CGAGC-GCTCGAGGGAACGTATAATTAAAGTAGTG 3'                 (SEQ ID NO:67))

and B

    (5' GCGCGGTACCAAGCTTC-AATTCGAGATAATACCC 3'                 (SEQ ID NO:68)).

The 350 nucleotide product was purified by gel electrophoresis usingGeneClean II (Bio101) and was cloned directly into the PCRII vector bysingle nucleotide overlap TA cloning (InVitrogen). Recombinant cloneswere characterized by restriction enzyme mapping and bydideoxynucleotide sequencing. Recombinant clones contained a novel XhoIsite 5' to the authentic GPA1 sequence and a novel KpnI site 3' to theauthentic GPA1 sequence donated respectively by primer A and primer B.

The NotI and SacI sites in the polylinker of Cadus 1013 (pRS414) wereremoved by restriction with these enzymes followed by filling in withthe Klenow fragment of DNA polymerase I and blunt end ligation to yieldCadus 1092. The 1.4 kb PstI-EcoRI 5' fragment of GPA1 fromYCplac111-GPA1 containing the GPA1 promoter and 5' untranslated regionof GPA1 was purified by gel electrophoresis using GeneClean (Bio101) andcloned into PstI-EcoRI restricted Cadus 1013 to yield Cadus 1087. ThePCR amplified XhoI-KpnI fragment encoding the 3' untranslated region ofGPA1 was excised from Cadus 1089 and cloned into XhoI KpnI restrictedCadus 1087 to yield Cadus 1092. The Not1 and Sacl sites in thepolylinker of Cadus 1092 were removed by restriction with these enzymes,filling in with the Klenow fragment of DNA polymerase I, and blunt endligation to yield Cadus 1110. The region of Cadus 1122 encoding theregion of GPA1 from the EcoRI site at -200 to +120 was amplified withVent DNA polymerase (New England Biolabs, Beverly, Mass.) with theprimers

    5' CCCGAATCCACCAATTTCTTTACG 3'                                                                       (SEQ ID NO:69)                                         and                                                                           5' GCGGCGTCGACGCGGCCGCGTAACAGT 3'                                                                    (SEQ ID NO:70).                                    

The amplified product, bearing an EcoRI site at its 5' end and novelSacI, NotI and SalI sites at its 3' end was restricted with EcoRI andSalI, gel purified using GeneClean II (Bio101), and cloned into EcoRIand SalI restricted Cadus 1110 to yield Cadus 1127. The DNA sequence ofthe vector between the EcoRI site at -200 and the KpnI site at the 3'end of the 3' untranslated region was verified by restriction enzymemapping and dideoxynucleotide DNA sequence analysis.

PCR amplification of GPA₄₁ -Gα proteins and cloning into Cadus 1127.cDNA clones encoding the human G alpha subunits Gαs,₋₋ Gαi2, Gαi3, andS. cerevisiae GPA1 were amplified with Vent thermostable polymerase (NewEngland Bioloabs, Beverly, Mass.). The primer pairs used in theamplification are as follows:

    GαS                                                                         Primer 1:                                                                            5'CTGCTGGAGCTCCGCCTGCTGCTGCTGGGTGCTGGAG3'                                                                     (SacI 5')                                                                              (SEQ ID NO:71)                Primer 2:                                                                            5'CTGCTGGTCGACGCGGCCGCGGGGGTTCCTTCTTAGAAGCAGC3'                                                               (SalI 3')                                                                              (SEQ ID NO:72)                Primer 3 :                                                                           5'GGGCTCGAGCCTTCTTAGAGCAGCTCGTAC3'                                                                            (XhoI 3')                                                                              (SEQ ID NO:73)            Gαi2                                                                        Primer 1 :                                                                           5'CTGCTGGAGCTCAAGTTGCTGCTGTTGGGTGCTGGGG3'                                                                     (SacI 5')                                                                              (SEQ ID NO:74)                Primer 2 :                                                                           5'CTGCTGGTCGACGCGGCCGCGCCCCTCAGAAGAGGCCGCGGTCC3'                                                              (SalI 3')                                                                              (SEQ ID NO:75)                Primer 3 :                                                                           5'GGGCTCGAGCCTCAGAAGAGGCCGCAGTC3'                                                                             (XhoI 3')                                                                              (SEQ ID NO:76)            Gαi3                                                                        Primer 1 :                                                                           5'CTGCTGGAGCTCAAGCTGCTGCTACTCGGTGCTGGAG3'                                                                     (SacI 5')                                                                              (SEQ ID NO:77)                Primer 2 :                                                                           5'CTGCTGGTCGACGCGGCCGCCACTAACATCCATGCTTCTCAATAAAGTC3'                                                         (SalI 3')                                                                              (SEQ ID NO:78)                Primer 3 :                                                                           5'GGGCTCGAGCATGCTTCTCAATAAAGTCCAC3'                                                                           (XhoI 3')                                                                              (SEQ ID NO:79)        

After amplification, products were purified by gel electrophoresis usingGeneClean II (Bio101) and were cleaved with the appropriate restrictionenzymes for cloning into Cadus 1127.

The hybrid GPA₄₁ -G.sub.α subunits were cloned via a SacI siteintroduced at the desired position near the 5' end of the amplifiedgenes and a SalI or XhoI site introduced in the 3' untranslated region.Ligation mixtures were electroporated into competent bacteria andplasmid DNA was prepared from 50 cultures of ampicillin resistantbacteria.

Construction of Integrating Vectors Encoding GPA₄₁ -G.sub.α Subunits.The coding region of each GPA₄₁ -Gα hybrid was cloned into anintegrating vector (pRS406=URA3 AmpR) using the BssHII sites flankingthe polylinker cloning sites in this plasmid. Cadus 1011 (pRS406) wasrestricted with BssHII, treated with shrimp alkaline phosphatase as perthe manufacturer's specifications, and the linearized vector waspurified by gel electrophoresis. Inserts from each of the GPA₄₁ -Gαhybrids were excised with BssHII from the parental plasmid, andsubcloned into gel purified Cadus 1011.

Construction of GPA_(Bam) -Gα Constructs. A novel BamHI site wasintroduced in frame into the GPA1 coding region by PCR amplificationusing Cadus 1179 (encoding a wildtype GPA1 allele with a novel NcoI siteat the initiator methionine) as the template, VENT polymerase, and thefollowing primers: Primer A=5' GCATCCATCAATAATCCAG 3' (SEQ ID NO:80) andPrimer B=5' GAAACAATGGA-TCCACTTCTTAC 3'. The 1.1 kb PCR product was gelpurified with GeneClean II (Bio101), restricted with NcoI and BamHI andcloned into NcoI-BamHI cut and phosphatased Cadus 1122 to yield Cadus1605. The sequence of Cadus 1605 was verified by restriction analysisand dideoxy-sequencing of double-stranded templates. RecombinantGPA_(Bam) -Gα hybrids of Gαs, Gαi2, and Gα16 were generated.Construction of Cadus 1855 encoding recombinant GPA_(Bam) -Gα₋₋ 16serves as a master example: construction of the other hybrids followedan analogous cloning strategy. The parental plasmid Cadus 1617, encodingnative Gα16, was restricted with NcoI and BamHI, treated with shrimpalkaline phosphatase as per the manufacturer's specifications and thelinearized vector was purified by gel electrophoresis. Cadus 1605 wasrestricted with NcoI and BamHI and the 1.1 kb fragment encoding theamino terminal 60% of GPA1 with a novel BamHI site at the 3' end wascloned into the NcoI- and BamHI-restricted Cadus 1617. The resultingplasmid encoding the GPA_(Bam) -Gα₋₋ 16 hybrid was verified byrestriction analysis and assayed in tester strains for an ability tocouple to yeast Gβγ and thereby suppress the gpa1 null phenotype. Twoadditional GPA_(Bam) -Gα₋₋ hybrids, GPA_(Bam) -Gαs₋₋ and GPA_(Bam)-Gαi2, described in this application were prepared in an analogousmanner using Cadus 1606 as the parental plasmid for the construction ofthe GPA_(Bam) -Gα₋₋ i2 hybrid and Cadus 1181 as the parental plasmid forthe construction of the GPA_(Bam) -Gαs hybrid.

Coupling by chimeric Gα₋₋ proteins.₋₋ The Gα chimeras described abovewere tested for the ability to couple a mammalian G protein-coupledreceptor to the pheromone response pathway in yeast. The results ofthese experiments are outlined in Table 5. Results obtained using GPA1₄₁-Gαi2 to couple the human C5a receptor to the pheromone response pathwayin autocrine strains of yeast are disclosed in Example 10 above.

                  TABLE 1                                                         ______________________________________                                        ABC TRANSPORTERS*                                                             Species       System      Substrate                                           ______________________________________                                        Bacteria                                                                      Salmonella    OppABCDF    Oligopeptides                                       typhimurium                                                                   Streptococcus AmiABCDEF   Oligopeptides                                       pneumoniae                                                                    Bacillus      Opp (SpoOK) Oligopeptides                                       subtilis                                                                      E. coli       Dpp         Dipeptides                                          Bacilus subtilis                                                                            DciA        Dipeptides                                          S. typhimurium                                                                              HisJQMP     Histidine                                           E. coli       HisJQMP     Histidine                                           E. coli       MalEFGK     Maltose                                             S. typhimurium                                                                              MalEFGK     Maltose                                             Enterobacter  MalEFGK     Maltose                                             aerogenes                                                                     E. coli       UgpABCE     sn-Glycerol-3-                                                                phosphate                                           E. coli       AraFGH      Arabinose                                           E. coli       RbsACD      Ribose                                              E. coli       GlnHPQ      Glutamine                                           S. typhimurium                                                                              ProU (VWX)  Glycine-betaine                                     E. coli       ProU (VWX)  Glycine-betaine                                     E. coli       LivHMGF (JK)                                                                              Leucine-                                                                      isoleucine-valine                                   E. coli       PstABC      Phosphate                                           Pseudomonas   NosDYF      Copper                                              stutzeri                                                                      E. coli       ChlJD       Molybdenum                                          E. coli       CysPTWAM    Sulphate-                                                                     Thiosulfate                                         E. coli       BtuCDE      Vitamin B12                                         E. coli       FhuBCD      Fe.sup.3+ -ferrichrome                              E. coli       FecBCDE     Fe.sup.3+ -dicitrate                                S. marcescens SfuABC      Fe.sup.3+                                           Mycoplasma    p37,29,69   ?                                                   E. coli       Phn/Psi     Alkyl-phosphonates                                                            (?)                                                 Streptomyces  DrrAB       Daunomycin/Doxorub                                  peucetius                 icin                                                Streptomyces  TlrC        Tylosin                                             fradiae                                                                       Staphylococcus                                                                              MsrA        Erythromycin                                                                  resistance                                          Agrobacterium OccJQMP     Octopine                                            tumefaciens                                                                   E. coli       HlyB        Haemolysin                                          Pasturella    LtkB        leukotoxin                                          E. coli       CvaB        Colicin V                                           Erwinia       PrtD        Proteases                                           chrysanthemi                                                                  Bordetella    CyaB        Cyclolysin                                          pertussis                                                                     Streptococcus ComA        Competence factor                                   pneumoniae                                                                    Rhizobium meliloti                                                                          NdvA        β-1,2-glucan                                   Agrobacterium ChvA        β-1,2-glucan                                   tumefaciens                                                                   Haemophilus   BexAB       Capsule                                             influenzae                polysaccharide                                      E. coli       KpsMT       Capsule                                                                       polysaccharide                                      Niesseria     CrtCD       Capsule                                                                       polysaccharide                                      E. coli       FtsE        Cell division                                       E. coli       UvrA        DNA repair                                          Rhizobium     NodI        Nodulation                                          leguminosarum                                                                 Rhizobium meliloti                                                                          OFR1        ?                                                   Cyanobacteria                                                                  Anabaena     HetA        Differentiation                                     Synchococcus  CysA        Sulphate                                            Yeast                                                                         S. cerevisiae STE6        a-mating peptide                                    S. cerevisiae ADP1        ?                                                   S. cerevisiae EF-3        Translation                                         Protozoa                                                                      Plasmodium    pfMDR       Chloroquine                                         Lieshmania    ltpgpA      Methotrexate/heavy                                                            metals                                              Insect                                                                        Drosophila    white-brown Eye pigments                                        Drosophila    Mdr49       Hydrophobic drugs?                                                Mdr65       ?                                                   Plants                                                                        Liverwort     MbpX        ?                                                   chloroplast                                                                   Animals                                                                       Man           CFTR        Chloride                                            Mouse         CFTR        Chloride                                            Xenopus       CFTR        Chloride                                            Cow           CFTR        Chloride                                            Dogfish       CFTR        Chloride                                            Man           MDR1        Hydrophobic drugs                                                 MDR3        ?                                                   Mouse         MDR1        Hydrophobic drugs                                                 MDR2        ?                                                                 MDR3        Hydrophobic drugs                                                 MDR3        Hydrophobic drugs                                   Hamster       Pgp1        Hydrophobic drugs                                                 Pgp2        Hydrophobic drugs                                                 Pgp3        ?                                                   Man           PMP70       Polypeptides?                                       Man           RING4-11    Peptides                                                          PSF1-PSF2   Peptides                                            Mouse         HAM1-HAM2   Peptides                                            Rat           Mtp1        Peptides                                            ______________________________________                                         *Adapted from Higgins, C. F. 1992. ABC Transporters: From Microorganizmz      to Man. Annu. Rev. Cell Biol. 8, 67-113.                                 

                  TABLE 2                                                         ______________________________________                                        HUMAN G PROTEIN-COUPLED SEVEN TRANSMEMBRANE                                   RECEPTORS: REFERENCES FOR CLONING                                             Receptor           Reference                                                  ______________________________________                                        α.sub.1A -adrenergic receptor                                                              Bruno et al. (1991)                                        α.sub.1B -adrenergic receptor                                                              Ramarao et al. (1992)                                      α.sub.2 -adrenergic receptor                                                               Lomasney et al. (1990)                                     α.sub.2B -adrenergic receptor                                                              Weinshank et al. (1990)                                    β.sub.1 -adrenergic receptor                                                                Frielle et al. (1987)                                      β.sub.2 -adrenergic receptor                                                                Kobilka et al. (1987)                                      β.sub.3 -adrenergic receptor                                                                Regan et al. (1988)                                        m.sub.1 AChR, m2 AChR, m.sub.3 AChR,                                                             Bonner et al. (1987)                                       m.sub.4 AChR       Peralta et al. (1987)                                      m5 AChR            Bonner et al. (1988)                                       D.sub.1 dopamine   Dearry et al. (1990)                                                          Zhou et al. (1990)                                                            Sunahara et al. (1990)                                                        Weinshank et al. (1991)                                    D.sub.2 dopamine   Grandy et al. (1989)                                       D.sub.3 dopamine   Sokoloff et al. (1990)                                     D.sub.4 dopamine   Van Tol et al. (1991)                                      D.sub.5 dopamine   M. Caron (unpub.)                                          A1 adenosine       Libert et al. (1992)                                                          Weinshank et al. (1991)                                    adenosine A2b      Pierce et al. (1992)                                       5-HT1a             Kobilka et al. (1987)                                                         Fargin et al. (1988)                                       5-HT1b             Hamblin et al. (1992)                                                         Mochizuki et al. (1992)                                    5HT1-like          Levy et al. (1992a)                                        5-HT1d             Levy et al. (1992b)                                        5HT1d-like         Hamblin and Metcalf (1991)                                 5HT1d beta         Demchyshyn et al. (1992)                                   substance K (neurokinin A)                                                                       Gerard et al. (1990)                                       substance P (NK1)  Gerard, et al. (1991);                                                        Takeda et al. (1991)                                       f-Met-Leu-Phe      Boulay et al. (1990)                                                          Murphy & McDermott (1991)                                                     DeNardin et al. (1992)                                     angiotensin II type 1                                                                            Furuta et al. (1992)                                       mas proto-oncogene Young et al. (1986)                                        endothelin ETA     Hayzer et al. (1992)                                                          Hosoda et al. (1991)                                       endothelin ETB     Nakamuta et al. (1991)                                                        Ogawa et al. (1991)                                        thrombin           Vu et al. (1991)                                           growth hormone-releasing                                                                         Mayo (1992)                                                hormone (GHRH)                                                                vasoactive intestinal                                                                            Sreedharan et al. (1991)                                   peptide (VIP)                                                                 oxytocin           Kimura et al., (1992)                                      somatostatin SSTR1 and                                                                           Yamada et al. (1992a)                                      SSTR2                                                                         SSTR3              Yamada et al. (1992b)                                      cannabinoid        Gerard et al. (1991)                                       follicle stimulating                                                                             Minegish et al. (1991)                                     hormone (FSH)                                                                 LH/CG              Minegish et al. (1990)                                     thyroid stimulating                                                                              Nagayama et al. (1989)                                     hormone (TSH)      Libert et al. (1989)                                                          Misrahi et al. (1990)                                      thromboxane A2     Hirata et al. (1991)                                       platelet-activating factor                                                                       Kunz et al. (1992)                                         (PAF)                                                                         C5a anaphylatoxin  Boulay et al. (1991)                                                          Gerard and Gerard (1991)                                   Interleukin 8 (IL-8) IL-                                                                         Holmes et al. (1991)                                       8RA                                                                           IL-8RB             Murphy and Tiffany (1991)                                  Delta Opioid       Evans et al. (1992)                                        Kappa Opioid       Xie et al. (1992)                                          mip-1/RANTES       Neote et al. (1993)                                                           Murphy et al., in press                                    Rhodopsin          Nathans and Hogness (1984)                                 Red opsin, Green opsin,                                                                          Nathans, et al. (1986)                                     Blue opsin                                                                    metabotropic glutamate                                                                           Tanabe et al. (1992)                                       mGluR1-6                                                                      histamine H2       Gantz et al. (1991)                                        ATP                Julius, David (unpub.)                                     neuropeptide Y     Herzog et al. (1992)                                                          Larhammar et al. (1992)                                    amyloid protein precursor                                                                        Kang et al. (1987)                                                            Mita, et al. (1988)                                                           Lemaire et al. (1989)                                      insulin-like growth factor                                                                       Kiess et al. (1988)                                        II                                                                            bradykinin         Hess et al. (1992)                                         gonadotropin-releasing                                                                           Chi et al. (1993)                                          hormone                                                                       cholecystokinin    Pisegna et al. (1992)                                      melanocyte stimulating                                                                           Chhajlane et al. (1992)                                    hormone receptor   Mountjoy et al. (1992)                                     antidiuretic hormone                                                                             Birnbaumer et al. (1992)                                   receptor                                                                      glucagon receptor  Sprecher et al. (1993)                                     adrenocorticotropic                                                                              Mountjoy et al. (1992)                                     hormone II                                                                    ______________________________________                                    

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                  TABLE 3                                                         ______________________________________                                        Detection of C5a production in yeast by ELISA.                                           R-L- R+L-     R-L+      R+L+                                       ______________________________________                                        [C5a] in culture                                                                           n.d.   n.d.     0.64 ng/ml                                                                            0.5 ng/ml                                                             =77 nM  =60 nM                                   [C5a] released                                                                             n.d.   n.d.     0.8 ng/ml                                                                             0.6 ng/ml                                from lysed cells*            =97 nM  =73 nM                                   ______________________________________                                         C5a was detected by enzymelinked immunosorbent assay (ELISA). Molar           concentrations were calculated using MW = 8273 as predicted by C5a            sequence.                                                                     *Determined by pelleting cells, resuspending cells in the original volume     breaking yeast with glass beads and assaying the resulting supernatant.       n.d. = not done                                                          

                  TABLE 4                                                         ______________________________________                                        Coupling of the C5a receptor to Gα chimeras in yeast.                             Expression                                                          Chimera   Context      Result                                                 ______________________________________                                        GPA1.sub.41 -Gαi2                                                                 single copy, Good signal to noise ratio:                                      integrated,  efficient coupling to yeast                                      GPA1 promoter                                                                              βγ.                                         GPA1.sub.41 -Gαi3                                                                 single copy, Poor signal to noise ratio:                                      integrated,  high background due to poor                                      GPA1 promoter                                                                              coupling to yeast βγ, high                                         LIRMA*.                                                GPA1βam-Gαi2                                                                 low copy plasmid,                                                                          Signal equal to that with                                        GPA 1 promoter                                                                             GPA1.sub.41 -Gαi2, however, back-                                       ground is greater.                                     GPA1βam-Gα16                                                                 low copy plasmid,                                                                          Poor signal to noise ratio,                                      GPA1 promoter                                                                              high background due to poor                                                   coupling to yeast βγ  high                                         LIRMA*.                                                GPA1βam-Gαs                                                                  low copy plasmid,                                                                          Unacceptably high background                                     GPA1 promoter                                                                              due to poor coupling to                                                       yeast β, high LIRMA*.                             ______________________________________                                         *LIRMA = Ligand Independent Receptor Mediated Activation. With this           phenomenon, there is an increase in growth on selective media for strains     containing heterologous receptor in the absence of ligand. It is possible     that some receptor antagonists would decrease LIRMA. It has been noted        (Milano, et al. 1994) that specific antagonists reduce LIRMA of the           β2 adrenergic receptor when that receptor is overexpressed in            transgenic mice.                                                              LIRMA may be exploited in several ways, including the identification of       antagonists capable of reducing the phenomenon. A subset of antagonists       would be expected to affect the receptor conformation in such a way as to     prevent the downstream signalling that occurs in the absence of agonist.      LIRMA can be exploited to identify new G proteincoupled receptors by          expressing cDNA clones in yeast strains expressing those chimeric G           proteins which couple only poorly to yeast βγ. In addition,  #     LIRMA may permit the identification of inhibitors that are specific for G     proteins.                                                                

                                      TABLE 5                                     __________________________________________________________________________    Sequence alignments of N-terminal regions of Gα subunits and            N-terminal sequences of GPA.sub.41 -Gα hybrid proteins.                 __________________________________________________________________________    A. Alignment of GPA1 with Gα Subunits                                   GPA1                                                                          MGC.TVSTOTIGDESDPFLONKRANDVIEOSLOLEKORDKNEIKLLLLGAGESGKSTVLKQLKLLHQ.....      GαS                                                                     MGCLGTS..KTEDQRNEEKAQREANKKIEKQLQKDKQVYRATHRLLLLGAGESGKSTIVKQMRILHV.....      Gαi2                                                                    MGC.TVS........AEDKAAAERSKMIDKNLREDGEKAAREVKLLLLGAGESGKSTIVKQMKIIHE.....      Gαi3                                                                    MGC.TVS........AEDKAAVERSKMIDRNLREDGEKAAKEVKLLLLGAGESGKSTIVKQMKIIHE.....      Gα16                                                                    MARSLTWRCCPWCLTEDEKAAARVDQEINRILLEQKKQDRGELKLLLLGPGESGKSTFIKQMRIIHG.....      B. GPA.sub.41 -Gα Hybrids                                               GPA.sub.41 - GαS                                                        MGC.TVSTOTIGDESDPFLONKRANDVIEOSLOLEKORDKNERKLLLLGAGESGKSTIVKQMRILHV.....      GPA.sub.41 - Gαi2                                                       MGC.TVSTOTIGDESDPFLONKRANDVIEOSLOLEKORDKNEVKLLLLGAGESGKSTIVKQMKIIHE.....      GPA.sub.41 - Gαi3                                                       MGC.TVSTOTIGDESDPFLONKRANDVIEOSLOLEKORDKNEVKLLLLGAGESGKSTIVKQMKIIHE.....      GPA.sub.41 - Gα16                                                       MGC.TVSTOTIGDESDPFLONKRANDVIEOSLOLEKORDKNELKLLLLGPGESGKSTFIKQMRIIHG.....      __________________________________________________________________________

                  TABLE 6                                                         ______________________________________                                        Coupling of Gα switch region hybrids to the                             pheromone response pathway.                                                            GPA1 amino   Gas amino acid                                          Protein  acid sequences                                                                             sequences   Phenotype                                   ______________________________________                                        GPA1     1-472        none        Couples with                                                                  Gβγ                              GαS                                                                              none         1-394       Couples with                                                                  Gβγ weakly                       GPA.sub.41 -S                                                                          1-41         42-394      Couples with                                                                  Gβγ weakly                       SGS      297-333      1-201 + 237-                                                                              Does not                                                          394         couple with                                                                   Gβγ                              GPA.sub.41 -SGS                                                                        1-41 + 297-333                                                                             42-201 + 237-                                                                             Couples with                                                      394         Gβγ weakly                       ______________________________________                                    

                                      TABLE 7                                     __________________________________________________________________________    Gα Subunit Alignment - "Switch Region"                                              β2        β3             α2                       __________________________________________________________________________                β4                                                           GPA1 RIDTTGITETEFNIGSSKFKVLDAGGQRSERKKWIHCFEGITAVLFVLAMSEYDQMLFEDER           GαS                                                                          .VL.S..F..K.QNDKVN.HMF.V....D......Q..NDV..II..V.S.S.NMVIR..NQ           Gαi2                                                                         .VK....V..H.TFKDLH..MF.V................V..II.CV.L.A..LV.ADE.M           Gαi3                                                                         .VK....V..H.TFKDLY..MF.V................V..II.CV.L.D..LV.A...E           GαO                                                                          .VK....V..H.TFKNLH.RLF.V...............DV..II.CN.L.G...V.H...T           Gα11                                                                         .VP....I.YP.DLENII..MV.................NV.SIM.LV.L.....C.E.NNQ           Gα16                                                                         .MP....N.YC.SVQKTNL.IV.................N.I.LIYLASL.....V.V.SDN           __________________________________________________________________________

                  TABLE 8                                                         ______________________________________                                        G protein-coupled receptors that couple through Gαi:                    ______________________________________                                        M2 muscarinic acetylcholine                                                   M4 muscarinic acetylcholine                                                   adenosine A1                                                                  adenosine A3                                                                  α.sub.2A -adrenergic                                                    α.sub.2B -adrenergic                                                    α.sub.2C -adrenergic                                                    bradykinin B.sub.2                                                            cannabinoid                                                                   D2 dopamine                                                                   D4 dopamine                                                                   ET.sub.B endothelin                                                           formyl-methionyl peptide receptor FPR.sub.1                                   metabotropic glutamateR.sub.2                                                 metabotropic glutamateR.sub.3                                                 metabotropic glutamateR.sub.4                                                 5HT.sub.1A (serotonin)                                                        5HT.sub.1B (serotonin)                                                        5HT.sub.1D (serotonin)                                                        5HT.sub.1E (serotonin)                                                        5HT.sub.1F (serotonin)                                                        neuropeptide Y                                                                delta opioid                                                                  prostaglandin EP3                                                             somatostatin 2                                                                somatostatin 3                                                                somatostatin 4                                                                thrombin                                                                      C5a                                                                           platelet activating factor                                                    angiotensin AT.sub.1                                                          angiotensin AT.sub.2                                                          IL-8                                                                          MCP1A                                                                         MCP1B                                                                         ______________________________________                                    

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    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 119                                           - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 89 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Met Arg Phe Pro Ser Ile Phe Thr Ala Val Le - #u Phe Ala Ala Ser Ser         #                15                                                           - Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Gl - #u Asp Glu Thr Ala Gln         #            30                                                               - Ile Pro Ala Glu Ala Val Ile Gly Tyr Leu As - #p Leu Glu Gly Asp Phe         #        45                                                                   - Asp Val Ala Val Leu Pro Phe Ser Asn Ser Th - #r Asn Asn Gly Leu Leu         #    60                                                                       - Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Al - #a Lys Glu Glu Gly Val         #80                                                                           - Ser Leu Asp Lys Arg Glu Ala Glu Ala                                                         85                                                            - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 76 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pr - #o Met Tyr Lys Arg Glu         #                15                                                           - Ala Glu Ala Glu Ala Trp His Trp Leu Gln Le - #u Lys Pro Gly Gln Pro         #            30                                                               - Met Tyr Lys Arg Glu Ala Asp Ala Glu Ala Tr - #p His Trp Leu Gln Leu         #        45                                                                   - Lys Pro Gly Gln Pro Met Tyr Lys Arg Glu Al - #a Asp Ala Glu Ala Trp         #    60                                                                       - His Trp Leu Gln Leu Lys Pro Gly Gln Pro Me - #t Tyr                         #75                                                                           - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 15 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 #    15                                                                       - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..24                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 #      37 GAA GGG GTA TCT TTG CTT AAGCTCGAGA TC - #T                          Lys Glu Glu Gly Val Ser Leu Leu                                                 1               5                                                           - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 8 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Lys Glu Glu Gly Val Ser Leu Leu                                               1               5                                                           - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 77 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - CGTGAAGCTT AAGCGTGAGG CAGAAGCTNN KNNKNNKNNK NNKNNKNNKN NK - #NNKNNKNN         60                                                                          #   77             G                                                          - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 19 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Lys Arg Glu Ala Glu Ala Xaa Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         #                15                                                           - Xaa Xaa Xaa                                                                 - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 36 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Met Gln Pro Ser Thr Ala Thr Ala Ala Pro Ly - #s Glu Lys Thr Ser Ser         #                15                                                           - Glu Lys Lys Asp Asn Tyr Ile Ile Lys Gly Va - #l Phe Trp Asp Pro Ala         #            30                                                               - Cys Val Ile Ala                                                                     35                                                                    - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 # 19               ATG                                                        - (2) INFORMATION FOR SEQ ID NO:10:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..27                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                #       36CCA AAA GAA AAG ACC TCG AGC TCGCTTAAG - #                           Ala Ala Pro Lys Glu Lys Thr Ser Ser                                             1               5                                                           - (2) INFORMATION FOR SEQ ID NO:11:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 9 amino                                                           (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                - Ala Ala Pro Lys Glu Lys Thr Ser Ser                                           1               5                                                           - (2) INFORMATION FOR SEQ ID NO:12:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 79 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: double                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                - GGTACTCGAG TGAAAAGAAG GACAACNNKN NKNNKNNKNN KNNKNNKNNK NN - #KNNKNNKT         60                                                                          # 79               ACG                                                        - (2) INFORMATION FOR SEQ ID NO:13:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 22 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                - Ser Ser Glu Lys Lys Asp Asn Xaa Xaa Xaa Xa - #a Xaa Xaa Xaa Xaa Xaa         Xaa                                                                           #                15                                                           - Xaa Cys Val Ile Ala                                                                 20                                                                    - (2) INFORMATION FOR SEQ ID NO:14:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                #        34        TAGT ATCAAAAATG TCTG                                       - (2) INFORMATION FOR SEQ ID NO:15:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                #       35         GTTT GAAAAAGTAA TTTCG                                      - (2) INFORMATION FOR SEQ ID NO:16:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:                                #             28   AATT TTCATGTC                                              - (2) INFORMATION FOR SEQ ID NO:17:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:                                #        34        GTGT CGCATTATAT TTAC                                       - (2) INFORMATION FOR SEQ ID NO:18:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:                                - CTAAAGAAGA AGGGGTATCT TTGCTTAAGC TCGAGATCTC GACTGATAAC AA - #CAGTGTAG         60                                                                          - (2) INFORMATION FOR SEQ ID NO:19:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:                                #          31      CTTT AAAAGAATGA G                                          - (2) INFORMATION FOR SEQ ID NO:20:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:                                #               25 GCAG AAGCT                                                 - (2) INFORMATION FOR SEQ ID NO:21:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 10 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (ix) FEATURE:                                                           #/note= The 3'-end of this sequence                                                          is linked - # to the 5'-end of SEQ ID NO:118 by (NNN)n         #is any chosen integer.                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:                                #        10                                                                   - (2) INFORMATION FOR SEQ ID NO:22:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:                                #   41             GCTT TCGAATAGAA ATGCAACCAT C                               - (2) INFORMATION FOR SEQ ID NO:23:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 59 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:                                - GCCGCTCCAA AAGAAAAGAC CTCGAGCTCG CTTAAGTTCT GCGTACAAAA AC - #GTTGTTC          59                                                                          - (2) INFORMATION FOR SEQ ID NO:24:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:                                #              26  GAAG GACAAC                                                - (2) INFORMATION FOR SEQ ID NO:25:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (ix) FEATURE:                                                           #/note= The 3'-end of this sequence                                                          is linked - # to the 5'-end of SEQ ID NO:119 by (NNN)n         #is any chosen integer.                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:                                # 20               CACA                                                       - (2) INFORMATION FOR SEQ ID NO:26:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:                                #             28   GAGG CAGAAGCT                                              - (2) INFORMATION FOR SEQ ID NO:27:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 57 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:                                - CGGATGATCA MNNMNNMNNM NNMNNMNNMN NMNNMNNMNN MNNMNNMNNA GC - #TTCTG            57                                                                          - (2) INFORMATION FOR SEQ ID NO:28:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:                                #              26  GAAG GACAAC                                                - (2) INFORMATION FOR SEQ ID NO:29:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 60 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:                                - CGTACTTAAG CAATAACACA MNNMNNMNNM NNMNNMNNMN NMNNMNNMNN MN - #NGTTGTCC         60                                                                          - (2) INFORMATION FOR SEQ ID NO:30:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:                                #        34        GATC GTGCTGCCAG CCGC                                       - (2) INFORMATION FOR SEQ ID NO:31:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 32 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:                                #          32      CTGC GCCGCTGCTG CC                                         - (2) INFORMATION FOR SEQ ID NO:32:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 36 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:                                #       36         AGGC AGAAGCTTGG TTGCAG                                     - (2) INFORMATION FOR SEQ ID NO:33:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:                                #             27   ATTG GTTGGCC                                               - (2) INFORMATION FOR SEQ ID NO:34:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 32 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:                                - Arg Asn Ser Ser Ser Ser Gly Ser Ser Gly Al - #a Gly Gln Lys Arg Glu         #                15                                                           - Ala Glu Ala Trp His Trp Leu Gln Leu Lys Pr - #o Gly Gln Pro Met Tyr         #            30                                                               - (2) INFORMATION FOR SEQ ID NO:35:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:                                #            29    CAAG AAGAAGCCG                                             - (2) INFORMATION FOR SEQ ID NO:36:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:                                #                24AGCG TGGG                                                  - (2) INFORMATION FOR SEQ ID NO:37:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:                                #             28   AGCT TATCGATA                                              - (2) INFORMATION FOR SEQ ID NO:38:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 28 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:                                #             28   ATAG CTATCTAG                                              - (2) INFORMATION FOR SEQ ID NO:39:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 71 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:                                - CTGGATGCGA AGACAGCTNN KNNKNNKNNK NNKNNKNNKN NKNNKNNKNN KN - #NKTGATCA         60                                                                          #       71                                                                    - (2) INFORMATION FOR SEQ ID NO:40:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 17 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:                                #   17             A                                                          - (2) INFORMATION FOR SEQ ID NO:41:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 56 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:                                - GCCGTCAGTA AAGCTTGGCA TTGGTTGCAG CCTATGTACT GATCAGTCTG TG - #ACGC             56                                                                          - (2) INFORMATION FOR SEQ ID NO:42:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:                                #     39T TGG TTG CAG CTA AAA CCT GGC CAA CC - #A ATG TAC                     Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pr - #o Met Tyr                       #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:43:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:                                - Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:44:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 20 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:                                # 20               AGCT                                                       - (2) INFORMATION FOR SEQ ID NO:45:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 69 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:                                - CGGATGATCA GTACATTGGT TGGCCAGGTT TTAGCTGCAA CCAATGCCAA GC - #TGAGTCTT         60                                                                          #         69                                                                  - (2) INFORMATION FOR SEQ ID NO:46:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:                                #     39T TGG CTA CAG CTA ACG CCT GGG CAA CC - #A ATG TAC                     Trp His Trp Leu Gln Leu Thr Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:47:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:                                - Trp His Trp Leu Gln Leu Thr Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:48:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:                                #     39T TGG CTG GAG CTT ATG CCT GGC CAA CC - #A TTA TAC                     Trp His Trp Leu Glu Leu Met Pro Gly Gln Pr - #o Leu Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:49:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:                                - Trp His Trp Leu Glu Leu Met Pro Gly Gln Pr - #o Leu Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:50:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:                                #     39T TGG ATG GAG CTA AGA CCT GGC CAA CC - #A ATG TAC                     Trp His Trp Met Glu Leu Arg Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:51:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:                                - Trp His Trp Met Glu Leu Arg Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:52:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:                                #         33G TTT GTT CAT TTT TTT GAT ATT CC - #G                             Tyr Ala Leu Phe Val His Phe Phe Asp Ile Pr - #o                               #     20                                                                      - (2) INFORMATION FOR SEQ ID NO:53:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:                                - Tyr Ala Leu Phe Val His Phe Phe Asp Ile Pr - #o                             #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:54:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:                                #         33T CAG GTG CGT TTT GTG GTT CTT GC - #T                             Phe Lys Gly Gln Val Arg Phe Val Val Leu Al - #a                               #             20                                                              - (2) INFORMATION FOR SEQ ID NO:55:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:                                - Phe Lys Gly Gln Val Arg Phe Val Val Leu Al - #a                             #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:56:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 33 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..33                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:                                #         33T CCG TCT TTT TTT TTT TTG CCT GC - #G                             Leu Met Ser Pro Ser Phe Phe Phe Leu Pro Al - #a                               #             20                                                              - (2) INFORMATION FOR SEQ ID NO:57:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:                                - Leu Met Ser Pro Ser Phe Phe Phe Leu Pro Al - #a                             #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:58:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:                                #             27   TTTC AACATGC                                               - (2) INFORMATION FOR SEQ ID NO:59:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:                                #                23ATCC CGC                                                   - (2) INFORMATION FOR SEQ ID NO:60:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 18 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:                                #  18              TG                                                         - (2) INFORMATION FOR SEQ ID NO:61:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:                                #              26  CTTT CTTAGG                                                - (2) INFORMATION FOR SEQ ID NO:62:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 35 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:                                #       35         CTAG AAGGAAGTGT TCACC                                      - (2) INFORMATION FOR SEQ ID NO:63:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 41 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:                                #   41             CATG GACTCCTTCA ATTATACCAC C                               - (2) INFORMATION FOR SEQ ID NO:64:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 42 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:                                #  42              CAGA AGCTACTCTG CAAAAGAAGA TC                              - (2) INFORMATION FOR SEQ ID NO:65:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:                                #            29    CGAG TTGCATGTC                                             - (2) INFORMATION FOR SEQ ID NO:66:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 38 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:66:                                #     38           AAAC CATGGGGTGT ACAGTGAG                                   - (2) INFORMATION FOR SEQ ID NO:67:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:67:                                #        34        CGTA TAATTAAAGT AGTG                                       - (2) INFORMATION FOR SEQ ID NO:68:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 34 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:68:                                #        34        CAAT TCGAGATAAT ACCC                                       - (2) INFORMATION FOR SEQ ID NO:69:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 24 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:69:                                #                24TCTT TACG                                                  - (2) INFORMATION FOR SEQ ID NO:70:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 27 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:70:                                #             27   CGCG TAACAGT                                               - (2) INFORMATION FOR SEQ ID NO:71:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:71:                                #      37          TGCT GCTGCTGGGT GCTGGAG                                    - (2) INFORMATION FOR SEQ ID NO:72:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 43 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:72:                                # 43               CCGC GGGGGTTCCT TCTTAGAAGC AGC                             - (2) INFORMATION FOR SEQ ID NO:73:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 30 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:73:                                #           30     AGAG CAGCTCGTAC                                            - (2) INFORMATION FOR SEQ ID NO:74:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:74:                                #      37          TGCT GCTGTTGGGT GCTGGGG                                    - (2) INFORMATION FOR SEQ ID NO:75:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 44 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:75:                                # 44               CCGC GCCCCTCAGA AGAGGCCGCG GTCC                            - (2) INFORMATION FOR SEQ ID NO:76:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 29 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:76:                                #            29    AGAG GCCGCAGTC                                             - (2) INFORMATION FOR SEQ ID NO:77:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 37 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:77:                                #      37          TGCT GCTACTCGGT GCTGGAG                                    - (2) INFORMATION FOR SEQ ID NO:78:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 49 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:78:                                #               49GCCGC CACTAACATC CATGCTTCTC AATAAAGTC                       - (2) INFORMATION FOR SEQ ID NO:79:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 31 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:79:                                #          31      CTCA ATAAAGTCCA C                                          - (2) INFORMATION FOR SEQ ID NO:80:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 19 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:80:                                # 19               CAG                                                        - (2) INFORMATION FOR SEQ ID NO:81:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 23 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:81:                                #                23TTCT TAC                                                   - (2) INFORMATION FOR SEQ ID NO:82:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 66 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:82:                                - Met Gly Cys Thr Val Ser Thr Gln Thr Ile Gl - #y Asp Glu Ser Asp Pro         #                15                                                           - Phe Leu Gln Asn Lys Arg Ala Asn Asp Val Il - #e Glu Gln Ser Leu Gln         #            30                                                               - Leu Glu Lys Gln Arg Asp Lys Asn Glu Ile Ly - #s Leu Leu Leu Leu Gly         #        45                                                                   - Ala Gly Glu Ser Gly Lys Ser Thr Val Leu Ly - #s Gln Leu Lys Leu Leu         #    60                                                                       - His Gln                                                                     65                                                                            - (2) INFORMATION FOR SEQ ID NO:83:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 65 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:83:                                - Met Gly Cys Leu Gly Thr Ser Lys Thr Glu As - #p Gln Arg Asn Glu Glu         #                15                                                           - Lys Ala Gln Arg Glu Ala Asn Lys Lys Ile Gl - #u Lys Gln Leu Gln Lys         #            30                                                               - Asp Lys Gln Val Tyr Arg Ala Thr His Arg Le - #u Leu Leu Leu Gly Ala         #        45                                                                   - Gly Glu Ser Gly Lys Ser Thr Ile Val Lys Gl - #n Met Arg Ile Leu His         #    60                                                                       - Val                                                                         65                                                                            - (2) INFORMATION FOR SEQ ID NO:84:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 58 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:84:                                - Met Gly Cys Thr Val Ser Ala Glu Asp Lys Al - #a Ala Ala Glu Arg Ser         #                15                                                           - Lys Met Ile Asp Lys Asn Leu Arg Glu Asp Gl - #y Glu Lys Ala Ala Arg         #            30                                                               - Glu Val Lys Leu Leu Leu Leu Gly Ala Gly Gl - #u Ser Gly Lys Ser Thr         #        45                                                                   - Ile Val Lys Gln Met Lys Ile Ile His Glu                                     #    55                                                                       - (2) INFORMATION FOR SEQ ID NO:85:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 58 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:85:                                - Met Gly Cys Thr Val Ser Ala Glu Asp Lys Al - #a Ala Val Glu Arg Ser         #                15                                                           - Lys Met Ile Asp Arg Asn Leu Arg Glu Asp Gl - #y Glu Lys Ala Ala Lys         #            30                                                               - Glu Val Lys Leu Leu Leu Leu Gly Ala Gly Gl - #u Ser Gly Lys Ser Thr         #        45                                                                   - Ile Val Lys Gln Met Lys Ile Ile His Glu                                     #    55                                                                       - (2) INFORMATION FOR SEQ ID NO:86:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 67 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:86:                                - Met Ala Arg Ser Leu Thr Trp Arg Cys Cys Pr - #o Trp Cys Leu Thr Glu         #                15                                                           - Asp Glu Lys Ala Ala Ala Arg Val Asp Gln Gl - #u Ile Asn Arg Ile Leu         #            30                                                               - Leu Glu Gln Lys Lys Gln Asp Arg Gly Glu Le - #u Lys Leu Leu Leu Leu         #        45                                                                   - Gly Pro Gly Glu Ser Gly Lys Ser Thr Phe Il - #e Lys Gln Met Arg Ile         #    60                                                                       - Ile His Gly                                                                 65                                                                            - (2) INFORMATION FOR SEQ ID NO:87:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 66 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:87:                                - Met Gly Cys Thr Val Ser Thr Gln Thr Ile Gl - #y Asp Glu Ser Asp Pro         #                15                                                           - Phe Leu Gln Asn Lys Arg Ala Asn Asp Val Il - #e Glu Gln Ser Leu Gln         #            30                                                               - Leu Glu Lys Gln Arg Asp Lys Asn Glu Arg Ly - #s Leu Leu Leu Leu Gly         #        45                                                                   - Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Ly - #s Gln Met Arg Ile Leu         #    60                                                                       - His Val                                                                     65                                                                            - (2) INFORMATION FOR SEQ ID NO:88:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 66 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:88:                                - Met Gly Cys Thr Val Ser Thr Gln Thr Ile Gl - #y Asp Glu Ser Asp Pro         #                15                                                           - Phe Leu Gln Asn Lys Arg Ala Asn Asp Val Il - #e Glu Gln Ser Leu Gln         #            30                                                               - Leu Glu Lys Gln Arg Asp Lys Asn Glu Val Ly - #s Leu Leu Leu Leu Gly         #        45                                                                   - Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Ly - #s Gln Met Lys Ile Ile         #    60                                                                       - His Glu                                                                     65                                                                            - (2) INFORMATION FOR SEQ ID NO:89:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 66 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:89:                                - Met Gly Cys Thr Val Ser Thr Gln Thr Ile Gl - #y Asp Glu Ser Asp Pro         #                15                                                           - Phe Leu Gln Asn Lys Arg Ala Asn Asp Val Il - #e Glu Gln Ser Leu Gln         #            30                                                               - Leu Glu Lys Gln Arg Asp Lys Asn Glu Val Ly - #s Leu Leu Leu Leu Gly         #        45                                                                   - Ala Gly Glu Ser Gly Lys Ser Thr Ile Val Ly - #s Gln Met Lys Ile Ile         #    60                                                                       - His Glu                                                                     65                                                                            - (2) INFORMATION FOR SEQ ID NO:90:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 66 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:90:                                - Met Gly Cys Thr Val Ser Thr Gln Thr Ile Gl - #y Asp Glu Ser Asp Pro         #                15                                                           - Phe Leu Gln Asn Lys Arg Ala Asn Asp Val Il - #e Glu Gln Ser Leu Gln         #            30                                                               - Leu Glu Lys Gln Arg Asp Lys Asn Glu Leu Ly - #s Leu Leu Leu Leu Gly         #        45                                                                   - Pro Gly Glu Ser Gly Lys Ser Thr Phe Ile Ly - #s Gln Met Arg Ile Ile         #    60                                                                       - His Gly                                                                     65                                                                            - (2) INFORMATION FOR SEQ ID NO:91:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:91:                                #     39T TGG TTG CAG CTA AAA CCT GGC CAG CC - #T ATG TAC                     Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pr - #o Met Tyr                       #             20                                                              - (2) INFORMATION FOR SEQ ID NO:92:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:92:                                - Trp His Trp Leu Gln Leu Lys Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:93:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:93:                                #     39T TGG TTG TCC TTG TCG CCC GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Ser Leu Ser Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:94:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:94:                                - Trp His Trp Leu Ser Leu Ser Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:95:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:95:                                #     39T TGG TTG TCC CTG GAC GCT GGC CAG CC - #T ATG TAC                     Trp His Trp Leu Ser Leu Asp Ala Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:96:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:96:                                - Trp His Trp Leu Ser Leu Asp Ala Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:97:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:97:                                #     39T TGG TTG ACC TTG ATG GCC GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Thr Leu Met Ala Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:98:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:98:                                - Trp His Trp Leu Thr Leu Met Ala Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:99:                                           -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:99:                                #     39T TGG TTG CAG CTG TCG GCG GGC CAG CC - #T ATG TAC                     Trp His Trp Leu Gln Leu Ser Ala Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:100:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:100:                               - Trp His Trp Leu Gln Leu Ser Ala Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:101:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:101:                               #     39T TGG TTG AGG TTG CAG TCC GGC CAG CC - #T ATG TAC                     Trp His Trp Leu Arg Leu Gln Ser Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:102:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:102:                               - Trp His Trp Leu Arg Leu Gln Ser Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:103:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:103:                               #     39T TGG TTG CGC TTG TCC GCC GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Arg Leu Ser Ala Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:104:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:104:                               - Trp His Trp Leu Arg Leu Ser Ala Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:105:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:105:                               #     39T TGG TTG TCG CTC GTC CCG GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Ser Leu Val Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:106:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:106:                               - Trp His Trp Leu Ser Leu Val Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:107:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:107:                               #     39T TGG TTG TCC CTG TAC CCC GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Ser Leu Tyr Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:108:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:108:                               - Trp His Trp Leu Ser Leu Tyr Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:109:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 39 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: cDNA                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: CDS                                                             (B) LOCATION: 1..39                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:109:                               #     39T TGG TTG CGG CTG CAG CCC GGG CAG CC - #T ATG TAC                     Trp His Trp Leu Arg Leu Gln Pro Gly Gln Pr - #o Met Tyr                       #     25                                                                      - (2) INFORMATION FOR SEQ ID NO:110:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 13 amino                                                          (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:110:                               - Trp His Trp Leu Arg Leu Gln Pro Gly Gln Pr - #o Met Tyr                     #                 10                                                          - (2) INFORMATION FOR SEQ ID NO:111:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:111:                               - Arg Ile Asp Thr Thr Gly Ile Thr Glu Thr Gl - #u Phe Asn Ile Gly Ser         #                15                                                           - Ser Lys Phe Lys Val Leu Asp Ala Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Gly Ile Thr Al - #a Val Leu Phe Val Leu         #        45                                                                   - Ala Met Ser Glu Tyr Asp Gln Met Leu Phe Gl - #u Asp Glu Arg                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:112:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:112:                               - Arg Val Leu Thr Ser Gly Ile Phe Glu Thr Ly - #s Phe Gln Asn Asp Lys         #                15                                                           - Val Asn Phe His Met Phe Asp Val Gly Gly Gl - #n Arg Asp Glu Arg Lys         #            30                                                               - Lys Trp Ile Gln Cys Phe Asn Asp Val Thr Al - #a Ile Ile Phe Val Val         #        45                                                                   - Ala Ser Ser Ser Tyr Asn Met Val Ile Arg Gl - #u Asp Asn Gln                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:113:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:113:                               - Arg Val Lys Thr Thr Gly Ile Val Glu Thr Hi - #s Phe Thr Phe Lys Asp         #                15                                                           - Leu His Phe Lys Met Phe Asp Val Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Gly Val Thr Al - #a Ile Ile Phe Cys Val         #        45                                                                   - Ala Leu Ser Ala Tyr Asp Leu Val Leu Ala As - #p Glu Glu Met                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:114:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:114:                               - Arg Val Lys Thr Thr Gly Ile Val Glu Thr Hi - #s Phe Thr Phe Lys Asp         #                15                                                           - Leu Tyr Phe Lys Met Phe Asp Val Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Gly Val Thr Al - #a Ile Ile Phe Cys Val         #        45                                                                   - Ala Leu Ser Asp Tyr Asp Leu Val Leu Ala Gl - #u Asp Glu Glu                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:115:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:115:                               - Arg Val Lys Thr Thr Gly Ile Val Glu Thr Hi - #s Phe Thr Phe Lys Asn         #                15                                                           - Leu His Phe Arg Leu Phe Asp Val Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Asp Val Thr Al - #a Ile Ile Phe Cys Asn         #        45                                                                   - Ala Leu Ser Gly Tyr Asp Gln Val Leu His Gl - #u Asp Glu Thr                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:116:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:116:                               - Arg Val Pro Thr Thr Gly Ile Ile Glu Tyr Pr - #o Phe Asp Leu Glu Asn         #                15                                                           - Ile Ile Phe Lys Met Val Asp Ala Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Asn Val Thr Se - #r Ile Met Phe Leu Val         #        45                                                                   - Ala Leu Ser Glu Tyr Asp Gln Cys Leu Glu Gl - #u Asn Asn Gln                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:117:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 62 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: peptide                                             -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:117:                               - Arg Met Pro Thr Thr Gly Ile Asn Glu Tyr Cy - #s Phe Ser Val Gln Lys         #                15                                                           - Thr Asn Leu Lys Ile Val Asp Ala Gly Gly Gl - #n Arg Ser Glu Arg Lys         #            30                                                               - Lys Trp Ile His Cys Phe Glu Asn Ile Ile Al - #a Leu Ile Tyr Leu Ala         #        45                                                                   - Ser Leu Ser Glu Tyr Asp Gln Val Leu Val Gl - #u Ser Asp Asn                 #    60                                                                       - (2) INFORMATION FOR SEQ ID NO:118:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 25 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (ix) FEATURE:                                                           #/note= The 5'-end of this sequence                                                          is linked - # to the 3'-end of SEQ ID NO:21 by (NNN)n          #is any chosen integer.                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:118:                               #               25 TTAA GTAGC                                                 - (2) INFORMATION FOR SEQ ID NO:119:                                          -      (i) SEQUENCE CHARACTERISTICS:                                          #pairs    (A) LENGTH: 26 base                                                           (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ii) MOLECULE TYPE: synthetic DNA                                       -     (ix) FEATURE:                                                           #/note= The 5'-end of this sequence                                                          is linked - # to the 3'-end of SEQ ID NO:25 by (NNN)n          #is any chosen integer.                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:119:                               #              26  CTCG AGTACC                                                __________________________________________________________________________

We claim:
 1. A mixture of recombinant yeast cells, each cell of whichhas a cell membrane and each cell of which comprises:(a) a heterologousG protein-coupled receptor expressed in the cell membrane of said cellsuch that signal transduction activity via said receptor is modulated byinteraction of an extracellular region of the receptor with anextracellular signal; and (b) a heterologous polypeptide, wherein theheterologous polypeptide is transported to a location allowinginteraction with the extracellular region of the receptor expressed inthe cell membrane; wherein collectively the mixture of cells expresses alibrary of said heterologous polypeptides, said library beingexpressible at a sufficient level such that modulation of the signaltransduction activity of the receptor by a heterologous polypeptidewithin the library provides a detectable signal.
 2. The yeast cells ofclaim 1, wherein the heterologous receptor is a G protein coupledreceptor that acts as a surrogate for an endogenous yeast pheromonereceptor in a pheromone system pathway of the yeast cells.
 3. The yeastcells of claim 2, which are a mutant strain of yeast cells having apheromone system pathway that is desensitized at slower rate relative toa wild type strain under the same conditions of continuous stimulationof the pheromone system pathway.
 4. The yeast cells of claim 2, whereina gene encoding the endogenous yeast pheromone receptor is notfunctionally expressed in the yeast cells.
 5. The yeast cells of claim2, wherein the yeast cells have a ste2 or ste3 mutation.
 6. The yeastcells of claim 2, wherein an endogenous pheromone gene is notfunctionally expressed in the yeast cells.
 7. The yeast cells of claim2, wherein an endogenous FAR1 gene is not functionally expressed in theyeast cells.
 8. The yeast cells of claim 2, wherein an endogenous SST2gene is not functionally expressed in the yeast cells.
 9. The yeastcells of claim 1 or 2, which further comprise a heterologous G proteinsubunit with which the heterologous receptor interacts.
 10. The yeastcells of claim 1 or 2, which further comprise a chimeric G proteinsubunit with which the heterologous receptor interacts.
 11. The yeastcells of claim 10, wherein the chimeric G protein subunit is a chimericGα subunit.
 12. The yeast cells of claim 2, which further comprises aselectable marker that is activated by the pheromone system pathway,thereby providing the detectable signal.
 13. The yeast cells of claim12, wherein the selectable marker comprises a pheromone-responsivepromoter operably linked to a selectable gene.
 14. The yeast cells ofclaim 13, wherein the pheromone-responsive promoter is the FUS1promoter.
 15. The yeast cells of claim 13, wherein the selectable geneis a HIS 3 gene.
 16. The yeast cells of claim 1, wherein theheterologous polypeptide includes a signal sequence that facilitatestransport of the polypeptide to a location allowing interaction with theextracellular region of the receptor.
 17. The yeast cells of claim 16,wherein the signal sequence corresponds to a leader peptide of theSaccharomyces cerevisiae α factor or a-factor.
 18. The yeast cells ofclaim 1, which belong to the species Saccharomyces cerevisiae.
 19. Theyeast cells of claim 1, wherein the library of heterologous polypeptidesincludes more than 1000 different polypeptide sequences.
 20. A mixtureof recombinant yeast cells, each cell of which has a cell membrane andeach cell of which comprises:(a) a heterologous G protein coupledreceptor (GPCR) expressed in the cell membrane of said cell such thatsignal transduction activity via said receptor is modulated byinteraction of an extracellular region of the receptor with anextracellular signal, said heterologous GPCR acting as a surrogate foran endogenous yeast pheromone receptor in a pheromone system pathway ofthe yeast cell; (b) a chimeric G protein subunit with which theheterologous receptor interacts; (c) a heterologous polypeptide, whereinthe heterologous polypeptide is transported to a location allowinginteraction with the extracellular region of the receptor expressed inthe cell membrane; and (d) a selectable marker that is activated by thepheromone system pathway; wherein collectively the mixture of cellsexpresses a library of said heterologous polypeptides, said librarybeing expressible at a sufficient level such that modulation of thesignal transduction activity of the receptor by a heterologouspolypeptide within the library provides a detectable signal mediated bythe selectable marker; and wherein said yeast cells have a mutation inat least one gene selected from the group consisting of FAR1, SST2,BAR1, SVG1, STE2, STE3, MFa1, MFa2, MFα1 and MFα2.
 21. The yeast cellsof claim 20, wherein the chimeric G protein subunit is a chimeric Gαsubunit.
 22. The yeast cells of claim 20, wherein the selectable markercomprises a pheromone-responsive promoter operably linked to aselectable gene.
 23. The yeast cells of claim 20, wherein theheterologous polypeptide includes a signal sequence that facilitatestransport of the polypeptide to a location allowing interaction with theextracellular region of the receptor.
 24. A method for identifying amodulator of a heterologous receptor protein expressed by a yeast cell,comprising:(i) providing a mixture of recombinant yeast cells, each cellof which has a cell membrane and comprises(a) a heterologous Gprotein-coupled receptor expressed in the cell membrane of said cellsuch that signal transduction activity via said receptor is modulated byinteraction of an extracellular region of the receptor with anextracellular signal; and (b) a heterologous test polypeptide, whereinthe heterologous test polypeptide is transported to a location allowinginteraction with the extracellular region of the receptor expressed inthe cell membrane; wherein collectively the mixture of cells expresses alibrary of said heterologous test polypeptides, said library beingexpressible at a sufficient level such that modulation of the signaltransduction activity of the receptor by a heterologous test polypeptidewithin the library provides a detectable signal (ii) allowing cellswithin the mixture to generate a detectable signal; and (iii)identifying the heterologous test polypeptide as a modulator of theheterologous receptor protein expressed by the yeast cell.
 25. Themethod of claim 24, wherein the heterologous receptor is a G proteincoupled receptor that acts as a surrogate for an endogenous yeastpheromone receptor in a pheromone system pathway of the yeast cells. 26.The method of claim 25, wherein the yeast cells are a mutant strain ofyeast cells having a pheromone system pathway that is desensitized atslower rate relative to a wild type strain under the same conditions ofcontinuous stimulation of the pheromone system pathway.
 27. The methodof claim 25, wherein a gene encoding the endogenous yeast pheromonereceptor is not functionally expressed in the yeast cells.
 28. Themethod of claim 25, wherein the yeast cells have a ste2 or ste3mutation.
 29. The method of claim 25, wherein an endogenous pheromonegene is not functionally expressed in the yeast cells.
 30. The method ofclaim 25, wherein an endogenous FAR1 gene is not functionally expressedin the yeast cells.
 31. The method of claim 25, wherein an endogenousSST2 gene is not functionally expressed in the yeast cells.
 32. Themethod of claim 24 or 25, wherein the yeast cells further comprise aheterologous G protein subunit with which the heterologous receptorinteracts.
 33. The method of claim 24 or 25, wherein the yeast cellsfurther comprise a chimeric G protein subunit with which theheterologous receptor interacts.
 34. The method of claim 33, wherein thechimeric G protein subunit is a chimeric Gα subunit.
 35. The method ofclaim 25, wherein the yeast cells further comprises a selectable markerthat is activated by the pheromone system pathway, thereby providing thedetectable signal.
 36. The method of claim 35, wherein the selectablemarker comprises a pheromone-responsive promoter operably linked to aselectable gene.
 37. The method of claim 36, wherein thepheromone-responsive promoter is the FUS1 promoter.
 38. The method ofclaim 36, wherein the selectable gene is a HIS 3 gene.
 39. The method ofclaim 24, wherein the heterologous polypeptide includes a signalsequence that facilitates transport of the polypeptide to a locationallowing interaction with the extracellular region of the receptor. 40.The method of claim 39 wherein the signal sequence corresponds to aleader peptide of the Saccharomyces cerevisiae α factor or a-factor. 41.The method of claim 24, wherein the yeast cells belong to the speciesSaccharomyces cerevisiae.
 42. The method of claim 24 wherein the libraryof heterologous polypeptides includes more than 1000 differentpolypeptide sequences.
 43. A method for identifying a modulator of aheterologous receptor protein expressed by a yeast cell, comprising:(i)providing a mixture of recombinant yeast cells, each cell of which has acell membrane and comprises:(a) a heterologous G protein coupledreceptor (GPCR) expressed in the cell membrane of said cell such thatsignal transduction activity via said receptor is modulated byinteraction of an extracellular region of the receptor with anextracellular signal, said heterologous GPCR acting as a surrogate foran endogenous yeast pheromone receptor in a pheromone system pathway ofthe yeast cell; (b) a chimeric G protein subunit with which theheterologous receptor interacts; (c) a heterologous polypeptide, whereinthe heterologous polypeptide is transported to a location allowinginteraction with the extracellular region of the receptor expressed inthe cell membrane; and (d) a selectable marker that is activated by thepheromone system pathway; wherein collectively the mixture of cellsexpresses a library of said heterologous polypeptides, said librarybeing expressible at a sufficient level such that modulation of thesignal transduction activity of the receptor by a heterologouspolypeptide within the library provides a detectable signal mediated bythe selectable marker; and wherein said yeast cells have a mutation inat least one gene selected from the group consisting of FAR1, SST2,BAR1, SVG1, STE2, STE3, MFa1, MFa2, MFα1 and MFα2; (ii) allowing cellswithin the mixture to generate a detectable signal; and (iii)identifying the heterologous test polypeptide as a modulator of theheterologous receptor protein expressed by the yeast cell.
 44. Themethod of claim 43, wherein the chimeric G protein subunit is a chimericGα subunit.
 45. The method of claim 43, wherein the selectable markercomprises a pheromone-responsive promoter operably linked to aselectable gene.
 46. The method of claim 43, wherein the heterologouspolypeptide includes a signal sequence that facilitates transport of thepolypeptide to a location allowing interaction with the extracellularregion of the receptor.
 47. A recombinant yeast cell which has a cellmembrane and which comprises:(a) a heterologous G protein-coupledreceptor expressed in the cell membrane of said cell such that signaltransduction activity via said receptor is modulated by interaction ofan extracellular region of the receptor with an extracellular signal;and (b) a heterologous polypeptide, wherein the heterologous polypeptideis transported to a location allowing interaction with the extracellularregion of the receptor expressed in the cell membrane; whereinmodulation of the signal transduction activity of the receptor by theheterologous polypeptide provides a detectable signal.
 48. A recombinantyeast cells which has a cell membrane and which comprises:(a) aheterologous G protein coupled receptor (GPCR) expressed in the cellmembrane of said cell such that signal transduction activity via saidreceptor is modulated by interaction of an extracellular region of thereceptor with an extracellular signal, said heterologous GPCR acting asa surrogate for an endogenous yeast pheromone receptor in a pheromonesystem pathway of the yeast cell; (b) a chimeric G protein subunit withwhich the heterologous receptor interacts; (c) a heterologouspolypeptide, wherein the heterologous polypeptide is transported to alocation allowing interaction with the extracellular region of thereceptor expressed in the cell membrane; and (d) a selectable markerthat is activated by the pheromone system pathway; wherein modulation ofthe signal transduction activity of the receptor by the heterologouspolypeptide provides a detectable signal mediated by the selectablemarker; and wherein the yeast cell has a mutation in at least one geneselected from the group consisting of FAR1, SST2, BAR1, SVG1, STE2,STE3, MFa1, MFa2, MFα1 and MFα2.